Peptides, devices, and methods for the detection of ehrlichia antibodies

ABSTRACT

The invention provides peptide compositions and mixtures useful for the detection of antibodies that bind to  Ehrlichia  antigens. The peptide compositions and mixtures comprise polypeptide sequences based on an immunogenic fragment of the  Ehrlichia  Outer Membrane Protein 1 (OMP-1) protein. The invention also provides devices, methods, and kits comprising such peptide compositions and mixtures useful for the detection of antibodies that bind to  Ehrlichia  antigens and the diagnosis of monocytic and/or granulocytic ehrlichiosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/712,578, filed Oct. 11, 2012, which is hereby incorporated byreference in its entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename: ABAX_(—)040_(—)01US_SeqList_ST25.txt, date recorded Oct. 10, 2013, file size 62kilobytes).

BACKGROUND OF THE INVENTION

Ehrlichia bacteria are obligate intracellular pathogens that infectcirculating lymphocytes in mammalian hosts. Ehrlichia canis andEhrlichia chaffeensis are members of the same sub-genus group thatinfect canines and humans and cause canine monocytic ehrlichiosis (CME)and human monocytic ehrlichiosis (HME), respectively. Another species ofEhrlichia known as Ehrlichia ewingii has tropism for granulocytes andcauses granulocytic ehrlichiosis. The canine disease is characterized byfever, epilepsy, incoordination, lethargy, bleeding episodes,lymphadenopathy, weight loss, and pancytopenia. In humans the disease ischaracterized by fever, headache, myalgia, and leukopenia. Earlydetection and treatment are important for treating both canine and humanehrlichiosis.

Indirect immunofluorescence assays (IFA) and enzyme-linked immunosorbentassays (ELISA) have typically been used in the diagnosis of thesediseases. These assays measure or otherwise detect the binding ofanti-Ehrlichia antibodies from a subject's blood, plasma, or serum toinfected cells, cell lysates, or partially purified whole Ehrlichiaproteins. However, currently known assays for detecting anti-Ehrlichiaantibodies or fragments thereof are severely limited in usefulnessbecause of sensitivity and specificity issues directly related to theimpure nature of the Ehrlichia antigen(s) used in these tests. That is,the currently known assays use mixtures of many whole Ehrlichia antigensor antigens that are not species specific.

Accordingly, there remains a need in the art for additional assays fordetecting Ehrlichia antigens and serodiagnosis of monocytic ehrlichiosisand granulocytic ehrlichiosis.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery that certainsequence variants in a fragment of the Ehrlichia Outer Membrane Protein1 (OMP-1) proteins provide for robust detection of an antibody responseagainst a range of Ehrlichia species. Accordingly, the inventionprovides compositions, devices, methods, and kits useful for thedetection of antibodies that bind to Ehrlichia antigens and thediagnosis of monocytic and/or granulocytic ehrlichiosis.

In one aspect, the invention provides peptides capable of binding toantibodies that recognize Ehrlichia antigens. In certain embodiments,peptides of the invention comprise a sequence ofS-X₂-K-E-D-K-Q-T-T-X₁₀-X₁₁-I-W-G-L-K-Q-X₁₈-W-X₂₀-G-X₂₂-P-X₂₄-X₂₅-X₂₆-X₂₇-X₂₈-X₂₉-X₃₀-X₃₁-X₃₂-X₃₃-X₃₄-X₃₅-X₃₆-X₃₇-X₃₈-X₃₉-C(SEQ ID NO: 1), or a fragment thereof, wherein X₂ is an amino acidselected from the group consisting of A and V, X₁₀ is an amino acidselected from the group consisting of T and V, X₁₁ is an amino acidselected from the group consisting of G and A, X₁₈ is an amino acidselected from the group consisting of E and Q, X₂₀ is an amino acidselected from the group consisting of D and N, X₂₂ is an amino acidselected from the group consisting of S and V, X₂₄ is an amino acidselected from the group consisting of A and I, X₂₅ is an amino acidselected from the group consisting of T and P, X₂₆ is an amino acidselected from the group consisting of S, N, and K, X₂₇ is any amino acidexcept H, N, S, or A, X₂₈ is any amino acid except A, S, or P, X₂₉ isany amino acid except D, P, N, or S, X₃₀ is any amino acid except A, E,D, or S, X₃₁ is any amino acid except D, N, V, or H, X₃₂ is any aminoacid except F or T, X₃₃ is any amino acid except N, F or I, X₃₄ is anyamino acid except N, T or D, X₃₅ is any amino acid except K, V or P, X₃₆is any amino acid except G, P, or S, X₃₇ is any amino acid except Y, N,or T, X₃₈ is any amino acid except S, Y, or I, and X₃₉ is any amino acidexcept F or S.

In some embodiments, peptides of the invention comprise a sequence ofS-X₂-K-E-D-K-Q-T-T-T-X₁₁-I-W-G-L-K-Q-X₁₈-W-D-G-X₂₂-P-X₂₄-X₂₅-X₂₆-X₂₇-X₂₈-X₂₉-X₃₀-X₃₁-X₃₂-X₃₃-X₃₄-X₃₅-X₃₆-X₃₇-X₃₈-X₃₉-C(SEQ ID NO: 83), or a fragment thereof, wherein X₂ is an amino acidselected from the group consisting of A and V, X₁₁ is an amino acidselected from the group consisting of G and A, X₁₈ is an amino acidselected from the group consisting of E and Q, X₂₂ is an amino acidselected from the group consisting of S and V, X₂₄ is an amino acidselected from the group consisting of A and I, X₂₅ is an amino acidselected from the group consisting of T and P, X₂₆ is an amino acidselected from the group consisting of S and N, X₂₇ is any amino acidexcept H, N, S, or A, X₂₈ is any amino acid except A, S, or P, X₂₉ isany amino acid except D, P, N, or S, X₃₀ is any amino acid except A, E,D, or S, X₃₁ is any amino acid except D, N, V, or R, X₃₂ is any aminoacid except F or T, X₃₃ is any amino acid except N, F or I, X₃₄ is anyamino acid except N, T or D, X₃₅ is any amino acid except K, V or P, X₃₆is any amino acid except G, P, or S, X₃₇ is any amino acid except Y, N,or T, X₃₈ is any amino acid except S, Y, or I, and X₃₉ is any amino acidexcept F or S.

In other embodiments, peptides of the invention comprise a sequence ofS-X₂-K-E-X₅-K-Q-X₈-T-X₁₀-X₁₁-X₁₂-X₁₃-G-L-K-Q-X₁₈-W-X₂₀-G-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-G-G-G-G-G-N-F-S-A-K-E-E-X₃₉-A-X₄₁-T-R-X₄₄-T-F-G-X₄₈-X₄₉-K-Q-Y-D-G-A-X₅₆-I-X₅₈-E-N-Q-V-Q-N-K-F-T-I-S-N-C(SEQ ID NO: 3) or a fragment thereof, wherein X₂ is an amino acidselected from the group consisting of A and V, X₅ is an amino acidselected from the group consisting of E and D, X₈ is an amino acidselected from the group consisting of T and P, X₁₀ is an amino acidselected from the group consisting of T and V, X₁₁ is an amino acidselected from the group consisting of G and A, X₁₂ is an amino acidselected from the group consisting of L and V, X₁₃ is an amino acidselected from the group consisting of Y and F, X₁₈ is an amino acidselected from the group consisting of D and N, X₂₀ is an amino acidselected from the group consisting of D and N, X₂₂ is an amino acidselected from the group consisting of S and V, X₂₃ is an amino acidselected from the group consisting of A, S, and T, X₂₄ is an amino acidselected from the group consisting of A and I, X₂₅ is an amino acidselected from the group consisting of T and P, X₂₆ is an amino acidselected from the group consisting of S, N, and K, X₃₉ is any aminoacid, X₄₁ is an amino acid selected from the group consisting of D andN, X₄₄ is any amino acid, X₄₈ is an amino acid selected from the groupconsisting of V and A, X₄₉ is any amino acid, X₅₆ is any amino acid, andX₅₈ is any amino acid.

In related embodiments, peptides of the invention comprise a sequence ofS-X₂-K-E-X₅-K-Q-X₈-T-X₁₀-X₁₁-X₁₂-X₁₃-G-L-K-Q-X₁₈-W-X₂₀-G-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-G-G-G-G-G-N-F-S-A-K-E-E-K-X₄₀-A-D-T-R-X₄₅-T-F-G-L-X₅₀-K-Q-T-D-G-A-X₅₇-I-X₅₉-E-N-Q-V-Q-N-K-F-T-I-S-N-C(SEQ ID NO: 85) or a fragment thereof, wherein X₂ is an amino acidselected from the group consisting of A and V, X₅ is an amino acidselected from the group consisting of E and D, X₈ is an amino acidselected from the group consisting of T and P, X₁₀ is an amino acidselected from the group consisting of T and V, X₁₁ is an amino acidselected from the group consisting of G and A, X₁₂ is an amino acidselected from the group consisting of L and V, X₁₃ is an amino acidselected from the group consisting of Y and F, X₁₈ is an amino acidselected from the group consisting of D and N, X₂₀ is an amino acidselected from the group consisting of D and N, X₂₂ is an amino acidselected from the group consisting of S and V, X₂₃ is an amino acidselected from the group consisting of A, S, and T, X₂₄ is an amino acidselected from the group consisting of A and I, X₂₅ is an amino acidselected from the group consisting of T and P, X₂₆ is an amino acidselected from the group consisting of S, N, and K, X₄₀ is any aminoacid, X₄₅ is any amino acid, X₅₀ is any amino acid, X₅₇ is any aminoacid, and X₅₉ is any amino acid.

In still other embodiments, peptides of the invention comprise asequence ofS-X₂-K-E-X₅-K-Q-X₈-T-X₁₀-X₁₁-X₁₂-X₁₃-G-L-K-Q-X₁₈-W-X₂₀-G-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-G-G-G-G-G-N-F-S-A-K-E-E-X₃₉-A-E-T-R-X₄₄-T-F-G-L-X₄₉-K-Q-Y-D-G-A-X₅₆-I-X₅₈E-N-Q-V-Q-N-K-F-T-I-S-N-C(SEQID NO: 72) or a fragment thereof, wherein X₂ is an amino acid selectedfrom the group consisting of A and V, X₅ is an amino acid selected fromthe group consisting of E and D, X₈ is an amino acid selected from thegroup consisting of T and P, X₁₀ is an amino acid selected from thegroup consisting of T and V, X₁₁ is an amino acid selected from thegroup consisting of G and A, X₁₂ is an amino acid selected from thegroup consisting of L and V, X₁₃ is an amino acid selected from thegroup consisting of Y and F, X₁₈ is an amino acid selected from thegroup consisting of D and N, X₂₀ is an amino acid selected from thegroup consisting of D and N, X₂₂ is an amino acid selected from thegroup consisting of S and V, X₂₃ is an amino acid selected from thegroup consisting of A, S, and T, X₂₄ is an amino acid selected from thegroup consisting of A and I, X₂₅ is an amino acid selected from thegroup consisting of T and P, X₂₆ is an amino acid selected from thegroup consisting of S, N, and K, X₃₉ is any amino acid, X₄₄ is any aminoacid, X₄₉ is any amino acid, X₅₆ is any amino acid, and X₅₈ is any aminoacid.

In certain embodiments, peptides of the invention comprise or consist ofa sequence of SEQ ID NO: 3 or SEQ ID NO: 72, wherein X₃₉ is K. In otherembodiments, peptides of the invention comprise or consist of a sequenceof SEQ ID NO: 3 or SEQ ID NO: 72, wherein X₄₄ is an amino acid selectedfrom the group consisting of K and R and X₄₉ is an amino acid selectedfrom the group consisting of E and D. In still other embodiments,peptides of the invention comprise or consist of a sequence of SEQ IDNO: 3 or SEQ ID NO: 72, wherein X₅₆ is an amino acid selected from thegroup consisting of K and Q and X₅₈ is an amino acid selected from thegroup consisting of E and T.

In some embodiments, peptides of the invention comprise a fragment ofSEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 72. Such fragments may compriseat least 10, 15, 20, 25, 30, or 35 contiguous amino acids from SEQ IDNO: 1 or at least 10, 15, 20, 25, 30, 35, or 40 contiguous amino acidsfrom SEQ ID NO: 3 or SEQ ID NO: 72. In certain embodiments, a peptide ofthe invention may comprise or consist of a sequence of SEQ ID NO: 5, SEQID NO: 6, SEQ ID NO: 71, SEQ ID NO: 84, or SEQ ID NO: 86.

In certain embodiments, peptides of the invention described herein mayfurther comprise an additional N-terminal peptide sequence. Theadditional N-terminal peptide sequence can comprise 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more amino acids and can be either a native or non-nativesequence. In other embodiments, peptides of the invention describedherein may further comprise an additional C-terminal sequence. Theadditional C-terminal peptide sequence can comprise 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more amino acids and can be either a native or non-nativesequence. In some embodiments, the non-native sequence comprises anon-OMP-1 Ehrlichia antigen (e.g., Ehrlichia p38, p43, p120, p140, p153,p156, p200, gp19, gp36, gp47, gp200, or HGE-3).

In certain embodiments, peptides of the invention comprise at least 25,30, 35, 40, 45, 50, or more amino acids. In certain embodiments,peptides of the invention are isolated (e.g., synthetic and/or purified)peptides. In certain embodiments, peptides of the invention areconjugated to a ligand. For example, in certain embodiments, thepeptides are biotinylated. In other embodiments, the peptides areconjugated to streptavidin, avidin, or neutravidin. In otherembodiments, the peptides are conjugated to a carrier protein (e.g.,serum albumin, keyhole limpet hemocyanin (KLH), or an immunoglobulin Fcdomain). In still other embodiments, the peptides are conjugated to adendrimer and/or are part of a multiple antigenic peptides system(MAPS).

In certain embodiments, peptides of the invention are attached to orimmobilized on a solid support. In one embodiment, the peptides of theinvention are attached to a solid support through a metallic nanolayer.In certain embodiments, the solid support is a bead (e.g., a colloidalparticle, metallic nanoparticle or nanoshell, latex bead, etc.), a flowpath in a lateral flow immunoassay device (e.g., a porous membrane), ablot (Western blot, a slot blot, or dot blot), a flow path in ananalytical or centrifugal rotor, or a tube or well (e.g., in a platesuitable for an ELISA assay).

In another aspect, the invention provides compositions comprising two ormore peptides of the invention. For example, in certain embodiments, thecomposition comprises a mixture or population of two, three, four, ormore different peptides of the invention, wherein each peptide comprisesa sequence of SEQ ID NO: 1. In some embodiments, the compositioncomprises a mixture or population of two, three, four, or more differentpeptides of the invention, wherein each peptide comprises a sequence ofSEQ ID NO: 3. In other embodiments, the composition comprises a mixtureor population of two, three, four, or more different peptides of theinvention, wherein each peptide comprises a sequence of SEQ ID NO: 72.In yet other embodiments, the composition comprises a mixture orpopulation of two, three, four, or more different peptides of theinvention, wherein each peptide comprises a sequence of SEQ ID NO: 7. Instill other embodiments, the composition comprises a mixture orpopulation of two, three, four, or more different peptides of theinvention, wherein each peptide comprises a sequence of SEQ ID NO: 70.In some embodiments, the composition comprises a mixture or populationof two, three, four, or more different peptides of the invention,wherein each peptide comprises a sequence of SEQ ID NO: 83. In otherembodiments, the composition comprises a mixture or population of two,three, four, or more different peptides of the invention, wherein eachpeptide comprises a sequence of SEQ ID NO: 85. In certain embodiments,the composition comprises a mixture or population of two, three, four,or more different peptides of the invention, wherein each peptidecomprises a sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 72, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86.

In another aspect, the invention provides nucleic acids comprising asequence encoding a peptide of the invention. In addition, the inventionprovides vectors comprising such nucleic acids, and host cellscomprising such vectors. In certain embodiments, the vector is a shuttlevector. In other embodiments, the vector is an expression vector (e.g.,a bacterial or eukaryotic expression vector). In certain embodiments,the host cell is a bacterial cell. In other embodiments, the host cellis a eukaryotic cell.

In another aspect, the invention provides devices. In certainembodiments, the devices are useful for performing an immunoassay. Forexample, in certain embodiments, the device is a lateral flowimmunoassay device. In other embodiments, the device is an analytical orcentrifugal rotor. In other embodiments, the device is a tube or a well,e.g., in a plate suitable for an ELISA assay. In still otherembodiments, the device is an electrochemical, optical, oropto-electronic sensor.

In certain embodiments, the device comprises a peptide of the invention.In other embodiments, the device comprises a mixture of differentpeptides of the invention. For example, in certain embodiments, thedevice comprises two, three, four, or more different peptides of theinvention. In certain embodiments, the peptide or each peptide in themixture comprises a sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:5, SEQ ID NO: 7, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 83, SEQ ID NO:84, SEQ ID NO: 85, or SEQ ID NO: 86. In certain embodiments, thepeptides are attached to or immobilized upon the device.

In another aspect, the invention provides methods of detecting in asample an antibody to an epitope of an Ehrlichia antigen. In certainembodiments, the methods comprise contacting a sample with a peptide ofthe invention, and detecting formation of an antibody-peptide complexcomprising said peptide, wherein formation of said complex is indicativeof the presence of an antibody to an epitope of a Ehrlichia antigen insaid sample. In certain embodiments, the Ehrlichia antigen is from aninfectious Ehrlichia species, such as Ehrlichia canis, Ehrlichiachaffeensis, Ehrlichia ewingii, or Ehrlichia muris. In certainembodiments, the methods comprise contacting the sample with a mixtureor population of different peptides of the invention (i.e. a mixture oftwo, three, four, or more different peptides of the invention). In someembodiments, the methods provide for detection of antibodies to antigensfrom multiple Ehrlichia species (e.g., Ehrlichia canis, Ehrlichiachaffeensis, Ehrlichia ewingii, and Ehrlichia muris) in a samplesimultaneously.

In certain embodiments, the peptide or each peptide in the mixture is anisolated (e.g., synthetic and/or purified) peptide. In certainembodiments, the peptide or mixture or population of peptides isattached to or immobilized upon a solid support. In one embodiment, thepeptide or mixture or population of peptides is attached to the solidsupport through a metallic (e.g., gold) nanolayer. In certainembodiments, the solid support is a bead or plurality of beads (e.g., acolloidal particle, a metallic nanoparticle or nanoshell, a latex bead,etc.), a flow path in a lateral flow immunoassay device (e.g., a porousmembrane), a flow path in an analytical or centrifugal rotor, a blot(Western blot, a slot blot, or dot blot), or a tube or a well (e.g., ina plate suitable for an ELISA assay). In certain embodiments, the solidsupport comprises metal, glass, a cellulose-based material (e.g.,nitrocellulose), or a polymer (e.g., polystyrene, polyethylene,polypropylene, polyester, nylon, polysulfone, etc.). In certainembodiments, the peptide or mixture or population of different peptidesis attached to a dendrimer and/or incorporated into a MAPS system. Incertain other embodiments, the peptide or mixture or population ofdifferent peptides is attached to BSA.

In certain embodiments, the detecting step comprises performing an ELISAassay. In other embodiments, the detecting step comprises performing alateral flow immunoassay. In other embodiments, the detecting stepcomprises performing an agglutination assay. In other embodiments, thedetecting step comprises spinning the sample in an analytical orcentrifugal rotor. In other embodiments, the detecting step comprisesanalyzing the sample using a Western blot, a slot blot, or a dot blot.In still other embodiments, the detecting step comprises analyzing thesample with an electrochemical sensor, an optical sensor, or anopto-electronic sensor. In certain embodiments, the detecting stepcomprises performing a wavelength shift assay.

In certain embodiments, the sample is a bodily fluid, such as blood,serum, plasma, cerebral spinal fluid, urine, mucus, or saliva. In otherembodiments, the sample is a tissue (e.g., a tissue homogenate) or acell lysate. In certain embodiments, the sample is from a wild animal(e.g., a deer or rodent, such as a mouse, chipmunk, squirrel, etc.). Inother embodiments, the sample is from a lab animal (e.g., a mouse, rat,guinea pig, rabbit, monkey, primate, etc.). In other embodiments, thesample is from a domesticated or feral animal (e.g., a dog, a cat, ahorse). In still other embodiments, the sample is from a human.

In another aspect, the invention provides methods of diagnosingmonocytic and/or granulocytic ehrlichiosis in a subject. In certainembodiments, the methods comprise contacting a sample from the subjectwith a peptide of the invention, and detecting formation of anantibody-peptide complex comprising said peptide, wherein formation ofsaid complex is indicative of the subject having monocytic and/orgranulocytic ehrlichiosis. In certain embodiments, the methods comprisecontacting the sample with a mixture or population of different peptidesof the invention (i.e. a mixture of two, three, four, or more differentpeptides of the invention).

In yet another aspect, the invention provides kits. In certainembodiments, the kits comprise a peptide of the invention. In certainembodiments, the kits comprise two, three, four, or more differentpeptides of the invention. The peptides can comprise a sequence of SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 70, SEQID NO: 72, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO:86. In certain embodiments, the peptides are attached to or immobilizedon a solid support optionally through a metallic nanolayer. In certainembodiments, the solid support is a bead (e.g., a colloidal particle, ametallic nanoparticle or nanoshell, a latex bead, etc.), a flow path ina lateral flow immunoassay device, a flow path in an analytical orcentrifugal rotor, or a tube or a well (e.g., in a plate). In certainembodiments, the peptide or peptides are attached to a dendrimer and/orincorporated into a MAPS system. In certain other embodiments, thepeptide or mixture of different peptides is attached to BSA.

In certain embodiments, the kits further comprise a population of beadsor a plate (e.g., a plate suitable for an ELISA assay). In otherembodiments, the kits further comprise a device, such as a lateral flowimmunoassay device, an analytical or centrifugal rotor, a Western blot,a dot blot, a slot blot, an electrochemical sensor, an optical sensor,or an opto-electronic sensor. In certain embodiments, the population ofbeads, the plate, or the device is useful for performing an immunoassay.For example, in certain embodiments, the population of beads, the plate,or the device is useful for detecting formation of an antibody-peptidecomplex comprising an antibody from a sample and a peptide of theinvention. In certain embodiments, a peptide or a mixture of differentpeptides of the invention is attached to or immobilized on the beads,the plate, or the device.

In certain embodiments, the kits further comprise an instruction. Forexample, in certain embodiments, the kits comprise an instructionindicating how to use a peptide of the invention to detect an antibodyto an Ehrlichia antigen or to diagnose monocytic and/or granulocyticehrlichiosis. In certain embodiments, the kits comprise an instructionindicating how to use a population of beads, a plate, or a device (e.g.,comprising a peptide or a mixture of different peptides of theinvention) to detect an antibody to one or more Ehrlichia antigens or todiagnose monocytic and/or granulocytic ehrlichiosis.

Additional aspects and embodiments of the invention will be apparentfrom the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of a double antigen sandwich assay which can be usedto detect antibodies to Ehrlichia antigens. In this embodiment, peptidesof the invention are immobilized to a suitable substrate (e.g.,nitrocellulose membrane, well of an ELISA plate) at a test site.Antibodies to Ehrlichia antigens in a test sample are bound by theimmobilized peptides of the invention. Test sample antibodies toappropriate Ehrlichia antigens will then bind to a second set ofpeptides of the invention that are conjugated to a detectable label(e.g., metallic nanoparticle or nanoshell (e.g., colloidal gold), horseradish peroxidase (HRP), alkaline phosphatase (ALP), fluorophore,colored latex particle), which detects the presence of the antibodiesbound to the first set of peptides immobilized at the test site. Incertain embodiments, to amplify the detection signal, protein A and/orprotein G molecules conjugated to a detectable label (e.g., metallicnanoparticle or nanoshell (e.g., colloidal gold), HRP, ALP, fluorophore,colored latex particle) may be applied to the test site where they willbind to the Fc region of any antibodies to Ehrlichia antigens capturedby the immobilized peptides of the invention.

FIG. 2 is a diagram of one type of indirect sandwich assay which can beused to detect antibodies to Ehrlichia antigens. In this embodiment,anti-human IgG/IgM, anti-dog IgG/IgM, or anti-cat IgG/IgM antibodies areimmobilized to a suitable substrate (e.g., nitrocellulose membrane, wellof an ELISA plate) at a test site. Antibodies to Ehrlichia antigens in atest sample are bound by the immobilized antibodies. Test sampleantibodies to appropriate Ehrlichia antigens will then bind to peptidesof the invention that are conjugated to a detectable label (e.g.,metallic nanoparticle or nanoshell (e.g., colloidal gold), HRP, ALP,fluorophore, colored latex particle).

FIG. 3 is a diagram of another type of indirect sandwich assay which canbe used to detect antibodies to Ehrlichia antigens. In this embodiment,peptides of the invention can be immobilized to a substrate (e.g.,nitrocellulose membrane, well of an ELISA plate) to captureanti-Ehrlichia antibodies in a test sample. Anti-human IgG/IgM, anti-dogIgG/IgM, or anti-cat IgG/IgM antibodies conjugated to a detectable label(e.g., metallic nanoparticle or nanoshell (e.g., colloidal gold), HRP,ALP, fluorophore, colored latex particle) can be used to detect thepresence of the antibodies bound to the immobilized peptides at the testsite.

FIG. 4 is a diagram of an immunoassay device which can be used to detectantibodies to Ehrlichia antigens. In this embodiment of an immunoassaydevice, peptides of the invention are immobilized to a suitablesubstrate (e.g., nitrocellulose membrane, well of an ELISA plate) at atest site. Anti-Ehrlichia antibodies in a test sample are bound by theimmobilized peptides of the invention. Protein A, Protein G, or aProtein A/G fusion protein conjugated to a detectable label (e.g.,metallic nanoparticle or nanoshell (e.g., colloidal gold), HRP, ALP,fluorophore, colored latex particle) is added to the system and binds tothe Fc portion of the captured anti-Ehrlichia antibody, therebyproducing a positive signal. In this embodiment, the device can furthercomprise a control site at which binding partners that recognize thedetectable label-conjugated protein A, detectable label-conjugatedprotein G, and/or detectable label-conjugated protein A/G fusion areimmobilized. Such binding partners may include, but are not limited to,anti-protein A, anti-protein G, mouse IgG, and/or other similar IgGmolecules.

FIG. 5 depicts one example of a lateral flow assay device that can beused to detect antibodies to Ehrlichia antigens. Peptides of theinvention are linked to a carrier protein (e.g. bovine serum albumin)and the resulting BSA-peptide conjugates are immobilized on anitrocellulose (NC) membrane at a test site (T). The same BSA-peptideconjugates are conjugated to a detectable label (e.g., colloidal gold)and deposited in a conjugate pad positioned upstream of the test site.Gold-conjugated protein A and gold-conjugated protein G (i.e. amplifier)is added to the conjugate pad to enhance the signal by binding to the Fcportion of the captured anti-Ehrlichia antibody. The device furthercomprises a control site (C) at which binding partners that recognizethe gold-conjugated protein A and/or gold-conjugated protein G areimmobilized.

DETAILED DESCRIPTION

As used herein, the following terms shall have the following meanings:

The term “antigen,” as used herein, refers to a molecule capable ofbeing recognized by an antibody. An antigen can be, for example, apeptide or a modified form thereof. An antigen can comprise one or moreepitopes.

The term “epitope,” as used herein, is a portion of an antigen that isspecifically recognized by an antibody. An epitope, for example, cancomprise or consist of a portion of a peptide (e.g., a peptide of theinvention). An epitope can be a linear epitope, sequential epitope, or aconformational epitope. In certain embodiments, epitopes may comprisenon-contiguous regions.

The term “OMP-1 protein” refers to any of the Outer Membrane Protein 1paralogs of Ehrlichia, including, but not limited to, E. canis P-30, E.canis P30-1, E. chaffeensis P28, E. chaffeensis OMP-1C, E. chaffeensisOMP-1D, E. chaffeensis OMP-1E, and E. chaffeensis OMP-1F.

The terms “nucleic acid,” “oligonucleotide” and “polynucleotide” areused interchangeably herein and encompass DNA, RNA, cDNA, whether singlestranded or double stranded, as well as chemical modifications thereof.

Single letter amino acid abbreviations used herein have their standardmeaning in the art, and all peptide sequences described herein arewritten according to convention, with the N-terminal end to the left andthe C-terminal end to the right.

Additional terms shall be defined, as required, in the detaileddescription that follows.

Compositions and Devices

The present invention is based, in part, on the discovery that certainsequence variants in a fragment of the Ehrlichia OMP-1 proteins providefor robust detection of an antibody response against a range ofEhrlichia species, including E. canis, E. chaffeensis, E. ewingii, andE. muris. Accordingly, in one aspect, the invention provides peptidescapable of binding to antibodies that recognize Ehrlichia antigens.

In certain embodiments, peptides of the invention comprise a sequence ofS-X₂-K-E-D-K-Q-T-T-X₁₀-X₁₁-I-W-G-L-K-Q-X₁₈-W-X₂₀-G-X₂₂-P-X₂₄-X₂₅-X₂₆-X₂₇-X₂₈-X₂₉-X₃₀-X₃₁-X₃₂-X₃₃-X₃₄-X₃₅-X₃₆-X₃₇-X₃₈-X₃₉-C(SEQ ID NO: 1), or a fragment thereof, wherein SEQ ID NO: 1, as usedthroughout the specification unless further specified, has the followingcharacteristics: X₂ is an amino acid selected from the group consistingof A and V; X₁₀ is an amino acid selected from the group consisting of Tand V; X₁₁ is an amino acid selected from the group consisting of G andA; X₁₈ is an amino acid selected from the group consisting of E and Q;X₂₀ is an amino acid selected from the group consisting of D and N; X₂₂is an amino acid selected from the group consisting of S and V; X₂₄ isan amino acid selected from the group consisting of A and I; X₂₅ is anamino acid selected from the group consisting of T and P; X₂₆ is anamino acid selected from the group consisting of S, N, and K; X₂₇ is anyamino acid except H, N, S, or A; X₂₈ is any amino acid except A, S, orP; X₂₉ is any amino acid except D, P, N, or S; X₃₀ is any amino acidexcept A, E, D, or S; X₃₁ is any amino acid except D, N, V, or H; X₃₂ isany amino acid except F or T; X₃₃ is any amino acid except N, F or I;X₃₄ is any amino acid except N, T or D; X₃₅ is any amino acid except K,V or P; X₃₆ is any amino acid except G, P, or S; X₃₇ is any amino acidexcept Y, N, or T; X₃₈ is any amino acid except S, Y, or I; and X₃₉ isany amino acid except F or S.

In certain embodiments, peptides of the invention comprise a sequence ofSEQ ID NO: 1, wherein X₂ is V and X₁₀ is T. In some embodiments,peptides of the invention comprise a sequence of SEQ ID NO: 1, whereinX₁₀ is T and X₂₆ is selected from the group consisting of S and N. Inother embodiments, peptides of the invention comprise a sequence of SEQID NO: 1, wherein X₂₄ is A, X₂₅ is selected from the group consisting ofT and P, and X₂₆ is selected from the group consisting of S and N. Instill other embodiments, peptides of the invention comprise a sequenceof SEQ ID NO: 1, wherein X₂₆ is selected from the group consisting of Sand N and X₃₁ is any amino acid except D, N, V, R, or H. In certainembodiments, peptides of the invention comprise a sequence of SEQ ID NO:1, wherein X₂₇-X₃₉ has a sequence selected from the group consisting ofQ-R-K-N-E-P-S-E-T-N-P-G-Q (SEQ ID NO: 74), M-V-E-F-E-E-L-Q-R-N-W-H-P(SEQ ID NO: 75), M-L-E-V-S-W-L-I-D-F-M-A-P (SEQ ID NO: 76), andQ-D-E-N-L-Y-S-S-I-F-F-V-P (SEQ ID NO: 77).

In related embodiments, peptides of the invention comprise a sequence ofS-X₂-K-E-D-K-Q-T-T-T-X₁₁-I-W-G-L-K-Q-X₁₈-W-D-G-X₂₂-P-X₂₄-X₂₅-X₂₆-X₂₇-X₂₈-X₂₉-X₃₀-X₃₁-X₃₂-X₃₃-X₃₄-X₃₅-X₃₆-X₃₇-X₃₈-X₃₉-C(SEQ ID NO: 83), or a fragment thereof, wherein X₂ is an amino acidselected from the group consisting of A and V, X₁₁ is an amino acidselected from the group consisting of G and A, X₁₈ is an amino acidselected from the group consisting of E and Q, X₂₂ is an amino acidselected from the group consisting of S and V, X₂₄ is an amino acidselected from the group consisting of A and I, X₂₅ is an amino acidselected from the group consisting of T and P, X₂₆ is an amino acidselected from the group consisting of S and N, X₂₇ is any amino acidexcept H, N, S, or A, X₂₈ is any amino acid except A, S, or P, X₂₉ isany amino acid except D, P, N, or S, X₃₀ is any amino acid except A, E,D, or S, X₃₁ is any amino acid except D, N, V, or R, X₃₂ is any aminoacid except F or T, X₃₃ is any amino acid except N, F or I, X₃₄ is anyamino acid except N, T or D, X₃₅ is any amino acid except K, V or P, X₃₆is any amino acid except G, P, or S, X₃₇ is any amino acid except Y, N,or T, X₃₈ is any amino acid except S, Y, or I, and X₃₉ is any amino acidexcept F or S. In some embodiments, peptides of the invention comprise asequence of SEQ ID NO: 83, wherein X₂₇-X₃₉ has a sequence selected fromthe group consisting of Q-R-K-N-E-P-S-E-T-N-P-G-Q (SEQ ID NO: 74),M-V-E-F-E-E-L-Q-R-N-W-H-P (SEQ ID NO: 75), M-L-E-V-S-W-L-I-D-F-M-A-P(SEQ ID NO: 76), and Q-D-E-N-L-Y-S-S-I-F-F-V-P (SEQ ID NO: 77).

In certain other embodiments of the invention, peptides of the inventioncomprise a sequence ofS-X₂-K-E-X₅-K-Q-X₈-T-X₁₀-X₁₁-X₁₂-X₁₃-G-L-K-Q-X₁₈-W-X₂₀-G-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-G-G-G-G-G-N-F-S-A-K-E-E-X₃₉-A-X₄₁-T-R-X₄₄-T-F-G-X₄₈-X₄₉-K-Q-Y-D-G-A-X₅₆-I-X₅₈-E-N-Q-V-Q-N-K-F-T-I-S-N-C(SEQ ID NO: 3) or a fragment thereof, wherein X₂ is an amino acidselected from the group consisting of A and V, X₅ is an amino acidselected from the group consisting of E and D, X₈ is an amino acidselected from the group consisting of T and P, X₁₀ is an amino acidselected from the group consisting of T and V, X₁₁ is an amino acidselected from the group consisting of G and A, X₁₂ is an amino acidselected from the group consisting of L and V, X₁₃ is an amino acidselected from the group consisting of Y and F, X₁₈ is an amino acidselected from the group consisting of D and N, X₂₀ is an amino acidselected from the group consisting of D and N, X₂₂ is an amino acidselected from the group consisting of S and V, X₂₃ is an amino acidselected from the group consisting of A, S, and T, X₂₄ is an amino acidselected from the group consisting of A and I, X₂₅ is an amino acidselected from the group consisting of T and P, X₂₆ is an amino acidselected from the group consisting of S, N, and K, X₃₉ is any aminoacid, X₄₁ is an amino acid selected from the group consisting of D andN, X₄₄ is any amino acid, X₄₈ is an amino acid selected from the groupconsisting of V and A, X₄₉ is any amino acid, X₅₆ is any amino acid, andX₅₈ is any amino acid.

In still other embodiments, peptides of the invention comprise asequence ofS-X₂-K-E-X₅-K-Q-X₈-T-X₁₀-X₁₁-X₁₂-X₁₃-G-L-K-Q-X₁₈-W-X₂₀-G-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-G-G-G-G-G-N-F-S-A-K-E-E-K-X₄₀-A-D-T-R-X₄₅-T-F-G-L-X₅₀-K-Q-T-D-G-A-X₅₇-I-X₅₉-E-N-Q-V-Q-N-K-F-T-I-S-N-C(SEQ ID NO: 85) or a fragment thereof, wherein X₂ is an amino acidselected from the group consisting of A and V, X₅ is an amino acidselected from the group consisting of E and D, X₈ is an amino acidselected from the group consisting of T and P, X₁₀ is an amino acidselected from the group consisting of T and V, X₁₁ is an amino acidselected from the group consisting of G and A, X₁₂ is an amino acidselected from the group consisting of L and V, X₁₃ is an amino acidselected from the group consisting of Y and F, X₁₈ is an amino acidselected from the group consisting of D and N, X₂₀ is an amino acidselected from the group consisting of D and N, X₂₂ is an amino acidselected from the group consisting of S and V, X₂₃ is an amino acidselected from the group consisting of A, S, and T, X₂₄ is an amino acidselected from the group consisting of A and I, X₂₅ is an amino acidselected from the group consisting of T and P, X₂₆ is an amino acidselected from the group consisting of S, N, and K, X₄₀ is any aminoacid, X₄₅ is any amino acid, X₅₀ is any amino acid, X₅₇ is any aminoacid, and X₅₉ is any amino acid.

In particular embodiments, peptides of the invention comprise a sequenceofS-X₂-K-E-X₅-K-Q-X₈-T-X₁₀-X₁₁-X₁₂-X₁₃-G-L-K-Q-X₁₈-W-X₂₀-G-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-G-G-G-G-G-N-F-S-A-K-E-E-X₃₉-A-E-T-R-X₄₄-T-F-G-L-X₄₉-K-Q-Y-D-G-A-X₅₆-I-X₅₈-E-N-Q-V-Q-N-K-F-T-I-S-N-C(SEQ ID NO: 72) or a fragment thereof, wherein X₂ is an amino acidselected from the group consisting of A and V, X₅ is an amino acidselected from the group consisting of E and D, X₈ is an amino acidselected from the group consisting of T and P, X₁₀ is an amino acidselected from the group consisting of T and V, X₁₁ is an amino acidselected from the group consisting of G and A, X₁₂ is an amino acidselected from the group consisting of L and V, X₁₃ is an amino acidselected from the group consisting of Y and F, X₁₈ is an amino acidselected from the group consisting of D and N, X₂₀ is an amino acidselected from the group consisting of D and N, X₂₂ is an amino acidselected from the group consisting of S and V, X₂₃ is an amino acidselected from the group consisting of A, S, and T, X₂₄ is an amino acidselected from the group consisting of A and I, X₂₅ is an amino acidselected from the group consisting of T and P, X₂₆ is an amino acidselected from the group consisting of S, N, and K, X₃₉ is any aminoacid, X₄₄ is any amino acid, X₄₉ is any amino acid, X₅₆ is any aminoacid, and X₅₈ is any amino acid. In one particular embodiment, peptidescomprising a sequence of SEQ ID NO: 72 enable the detection ofantibodies to Ehrlichia antigens from multiple species (e.g., E. canis,E. chaffeensis, E. ewingii, and E. muris) simultaneously.

In some embodiments, peptides of the invention comprise or consist of asequence of SEQ ID NO: 3 or SEQ ID NO: 72, wherein X₃₉ is K. In otherembodiments, peptides of the invention comprise or consist of a sequenceof SEQ ID NO: 3 or SEQ ID NO: 72, wherein X₄₄ is an amino acid selectedfrom the group consisting of K and R and X₄₉ is an amino acid selectedfrom the group consisting of E and D. In still other embodiments,peptides of the invention comprise or consist of a sequence of SEQ IDNO: 3 or SEQ ID NO: 72, wherein X₅₆ is an amino acid selected from thegroup consisting of K and Q and X₅₈ is an amino acid selected from thegroup consisting of E and T.

In another aspect of the invention, peptides of the invention compriseor consist of a sequence ofS-V-K-X₄-D-K-Q-X₈-T-X₁₀-V-L-W-G-I-R-Q-N-W-X₂₀-G-X₂₂-X₂₃-A-X₂₅-X₂₆-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 7), wherein X₄ is an amino acid selected from the groupconsisting of E and N, X₈ is an amino acid selected from the groupconsisting of P and S, X₁₀ is an amino acid selected from the groupconsisting of A and S, X₂₀ is an amino acid selected from the groupconsisting of E and Q, X₂₂ is an amino acid selected from the groupconsisting of P and T, X₂₃ is an amino acid selected from the groupconsisting of S and V, X₂₅ is an amino acid selected from the groupconsisting of T and P, and X₂₆ is an amino acid selected from the groupconsisting of S and N.

In certain embodiments, a peptide of the invention comprises or consistsof the sequenceS-V-K-E-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-Q-G-P-S-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 8);S-V-K-E-D-K-Q-S-T-A-V-L-W-G-I-R-Q-N-W-Q-G-P-S-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 9);S-V-K-E-D-K-Q-P-T-S-V-L-W-G-I-R-Q-N-W-Q-G-P-S-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 10);S-V-K-E-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-E-G-P-S-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 11);S-V-K-E-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-Q-G-T-S-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 12);S-V-K-E-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-Q-G-P-V-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 13);S-V-K-E-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-Q-G-P-S-A-P-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 14);S-V-K-E-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-Q-G-P-S-A-T-N-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 15);S-V-K-E-D-K-Q-S-T-S-V-L-W-G-I-R-Q-N-W-Q-G-P-S-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 16);S-V-K-E-D-K-Q-S-T-A-V-L-W-G-I-R-Q-N-W-E-G-P-S-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 17);S-V-K-E-D-K-Q-S-T-A-V-L-W-G-I-R-Q-N-W-Q-G-T-S-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 18);S-V-K-E-D-K-Q-S-T-A-V-L-W-G-I-R-Q-N-W-Q-G-P-V-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 19);S-V-K-E-D-K-Q-S-T-A-V-L-W-G-I-R-Q-N-W-Q-G-P-S-A-P-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 20);S-V-K-E-D-K-Q-S-T-A-V-L-W-G-I-R-Q-N-W-Q-G-P-S-A-P-N-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 21);S-V-K-E-D-K-Q-P-T-S-V-L-W-G-I-R-Q-N-W-E-G-P-S-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 22);S-V-K-E-D-K-Q-P-T-S-V-L-W-G-I-R-Q-N-W-Q-G-T-S-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 23);S-V-K-E-D-K-Q-P-T-S-V-L-W-G-I-R-Q-N-W-Q-G-P-V-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 24);S-V-K-E-D-K-Q-P-T-S-V-L-W-G-I-R-Q-N-W-Q-G-P-S-A-P-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 25);S-V-K-E-D-K-Q-P-T-S-V-L-W-G-I-R-Q-N-W-Q-G-P-S-A-T-N-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 26);S-V-K-E-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-E-G-T-S-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 27);S-V-K-E-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-E-G-P-V-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 28);S-V-K-E-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-E-G-P-S-A-P-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 29);S-V-K-E-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-E-G-P-S-A-T-N-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 30);S-V-K-E-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-Q-G-T-V-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 31);S-V-K-E-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-Q-G-T-S-A-P-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 32);S-V-K-E-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-Q-G-T-S-A-T-N-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 33);S-V-K-E-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-E-G-T-S-A-T-N-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 34);S-V-K-E-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-Q-G-P-V-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 35);S-V-K-E-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-Q-G-P-V-A-P-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 36);S-V-K-E-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-Q-G-P-V-A-T-N-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 37); orS-V-K-E-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-E-G-P-V-A-P-N-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 38).

In other embodiments, a peptide of the invention comprises or consistsof the sequenceS-V-K-N-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-Q-G-P-S-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 39);S-V-K-N-D-K-Q-S-T-A-V-L-W-G-I-R-Q-N-W-Q-G-P-S-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 40);S-V-K-N-D-K-Q-P-T-S-V-L-W-G-I-R-Q-N-W-Q-G-P-S-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 41);S-V-K-N-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-E-G-P-S-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 42);S-V-K-N-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-Q-G-T-S-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 43);S-V-K-N-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-Q-G-P-V-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 44);S-V-K-N-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-Q-G-P-S-A-P-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 45);S-V-K-N-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-Q-G-P-S-A-T-N-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 46);S-V-K-N-D-K-Q-S-T-S-V-L-W-G-I-R-Q-N-W-Q-G-P-S-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 47);S-V-K-N-D-K-Q-S-T-A-V-L-W-G-I-R-Q-N-W-E-G-P-S-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 48);S-V-K-N-D-K-Q-S-T-A-V-L-W-G-I-R-Q-N-W-Q-G-T-S-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 49);S-V-K-N-D-K-Q-S-T-A-V-L-W-G-I-R-Q-N-W-Q-G-P-V-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 50);S-V-K-N-D-K-Q-S-T-A-V-L-W-G-I-R-Q-N-W-Q-G-P-S-A-P-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 51);S-V-K-N-D-K-Q-S-T-A-V-L-W-G-I-R-Q-N-W-Q-G-P-S-A-P-N-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 52);S-V-K-N-D-K-Q-P-T-S-V-L-W-G-I-R-Q-N-W-E-G-P-S-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 53);S-V-K-N-D-K-Q-P-T-S-V-L-W-G-I-R-Q-N-W-Q-G-T-S-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 54);S-V-K-N-D-K-Q-P-T-S-V-L-W-G-I-R-Q-N-W-Q-G-P-V-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 55);S-V-K-N-D-K-Q-P-T-S-V-L-W-G-I-R-Q-N-W-Q-G-P-S-A-P-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 56);S-V-K-N-D-K-Q-P-T-S-V-L-W-G-I-R-Q-N-W-Q-G-P-S-A-T-N-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 57);S-V-K-N-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-E-G-T-S-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 58);S-V-K-N-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-E-G-P-V-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 59);S-V-K-N-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-E-G-P-S-A-P-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 60);S-V-K-N-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-E-G-P-S-A-T-N-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 61);S-V-K-N-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-Q-G-T-V-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 62);S-V-K-N-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-Q-G-T-S-A-P-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 63);S-V-K-N-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-Q-G-T-S-A-T-N-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 64);S-V-K-N-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-E-G-T-S-A-T-N-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 65);S-V-K-N-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-Q-G-P-V-A-T-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 66);S-V-K-N-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-Q-G-P-V-A-P-S-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 67);S-V-K-N-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-Q-G-P-V-A-T-N-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 68); orS-V-K-N-D-K-Q-P-T-A-V-L-W-G-I-R-Q-N-W-E-G-P-V-A-P-N-Q-V-E-V-E-W-Q-Q-R-G-W-G-G-C(SEQ ID NO: 69).

In one particular embodiment of the invention, peptides of the inventioncomprise or consist of a sequence ofS-X₂-K-D-X₅-K-Q-X₈-T-X₁₀-X₁₁-X₁₂-X₁₃-G-L-X₁₆-Q-X₁₈-X₁₉-X₂₀-G-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-X₂₇-X₂₈-X₂₉-X₃₀-X₃₁-X₃₂-X₃₃-X₃₄-X₃₅-X₃₆-X₃₇-X₃₈-X₃₉-C(SEQ ID NO: 70), wherein X₂ is an amino acid selected from the groupconsisting of A and V, X₅ is an amino acid selected from the groupconsisting of E and D, X₈ is an amino acid selected from the groupconsisting of T and P, X₁₀ is an amino acid selected from the groupconsisting of S, V, and A, X₁₁ is an amino acid selected from the groupconsisting of G and A, X₁₂ is an amino acid selected from the groupconsisting of L and V, X₁₃ is an amino acid selected from the groupconsisting of Y, F, and W, X₁₆ is an amino acid selected from the groupconsisting of K and R, X₁₈ is an amino acid selected from the groupconsisting of D and N, X₁₉ is an amino acid selected from the groupconsisting of W and F, X₂₀ is an amino acid selected from the groupconsisting of D and N, X₂₂ is an amino acid selected from the groupconsisting of T and V, X₂₃ is an amino acid selected from the groupconsisting of A, S, and T, X₂₄ is an amino acid selected from the groupconsisting of A and I, X₂₅ is an amino acid selected from the groupconsisting of T and P, X₂₆ is an amino acid selected from the groupconsisting of S, N, and K, and each of X₂₇-X₃₉ is any amino acid. Incertain embodiments, X₂₇-X₃₉ has a sequence selected from the groupconsisting of Q-R-K-N-E-P-S-E-T-N-P-G-Q (SEQ ID NO: 74),M-V-E-F-E-E-L-Q-R-N-W-H-P (SEQ ID NO: 75), M-L-E-V-S-W-L-I-D-F-M-A-P(SEQ ID NO: 76), Q-D-E-N-L-Y-S-S-I-F-F-V-P (SEQ ID NO: 77),Q-R-K-N-D-P-S-E-T-S-P-G-Q (SEQ ID NO: 78), M-A-P-F-H-E-L-D-V-N-N-H-P(SEQ ID NO: 79), S-L-N-V-S-F-L-I-D-P-M-A-P (SEQ ID NO: 80), andQ-D-S-N-L-Y-S-S-I-F-F-V-P (SEQ ID NO: 81).

In certain embodiments, peptides of the invention comprise a sequence ofSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 70, SEQ ID NO: 72,SEQ ID NO: 83, or SEQ ID NO: 85 and an additional N-terminal peptidesequence (e.g., an N-terminal extension). The additional N-terminalpeptide sequence can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 20, 25, or more amino acids. In certain embodiments, theN-terminal peptide sequence has a length of about 5 to about 10, about10 to about 15, about 15 to about 20, about 20 to about 25, about 25 toabout 30, about 30 to about 40, or about 40 to about 50 amino acids. Inone embodiment, the N-terminal peptide sequence can be one or morelinking residues (e.g. one or more glycine, cysteine, or serineresidues). For instance, in certain embodiments, the carboxyl-terminalcysteine residue in any of the sequences described herein can be locatedat the amino terminus instead. Accordingly, in some embodiments,peptides of the invention comprise or consist of a sequence ofC-S-X₃-K-E-D-K-Q-T-T-X₁₁-X₁₂-I-W-G-L-K-Q-X₁₉-W-X₂₁-G-X₂₃-P-X₂₅-X₂₆-X₂₇-X₂₈-X₂₉-X₃₀-X₃₁-X₃₂-X₃₃-X₃₄-X₃₅-X₃₆-X₃₇-X₃₈-X₃₉-X₄₀(SEQ ID NO: 2), wherein X₃ is an amino acid selected from the groupconsisting of A and V, X₁₁ is an amino acid selected from the groupconsisting of T and V, X₁₂ is an amino acid selected from the groupconsisting of G and A, X₁₉ is an amino acid selected from the groupconsisting of E and Q, X₂₁ is an amino acid selected from the groupconsisting of D and N, X₂₃ is an amino acid selected from the groupconsisting of S and V, X₂₅ is an amino acid selected from the groupconsisting of A and I, X₂₆ is an amino acid selected from the groupconsisting of T and P, X₂₇ is an amino acid selected from the groupconsisting of S, N, and K, X₂₈ is any amino acid except H, N, S, or A,X₂₉ is any amino acid except A, S, or P, X₃₀ is any amino acid except D,P, N, or S, X₃₁ is any amino acid except A, E, D, or S, X₃₂ is any aminoacid except D, N, V, or H, X₃₃ is any amino acid except F or T, X₃₄ isany amino acid except N, F or I, X₃₅ is any amino acid except N, T or D,X₃₆ is any amino acid except K, V or P, X₃₇ is any amino acid except G,P, or S, X₃₈ is any amino acid except Y, N, or T, X₃₉ is any amino acidexcept S, Y, or I, and X₄₀ is any amino acid except F or S. In stillother embodiments, peptides of the invention comprise or consist of asequence ofC-S-X₃-K-E-X₆-K-Q-X₉-T-X₁₁-X₁₂-X₁₃-X₁₄-G-L-K-Q-X₁₉-W-X₂₁-G-X₂₃-X₂₄-X₂₅-X₂₆-X₂₇-G-G-G-G-G-N-F-S-A-K-E-E-X₄₀-A-X₄₂-T-R-X₄₅-T-F-G-X₄₉-X₅₀-K-Q-Y-D-G-A-X₅₇-I-X₅₉-E-N-Q-V-Q-N-K-F-T-I-S-N(SEQ ID NO: 4), wherein X₃ is an amino acid selected from the groupconsisting of A and V, X₆ is an amino acid selected from the groupconsisting of E and D, X₉ is an amino acid selected from the groupconsisting of T and P, X₁₁ is an amino acid selected from the groupconsisting of T and V, X₁₂ is an amino acid selected from the groupconsisting of G and A, X₁₃ is an amino acid selected from the groupconsisting of L and V, X₁₄ is an amino acid selected from the groupconsisting of Y and F, X₁₉ is an amino acid selected from the groupconsisting of D and N, X₂₁ is an amino acid selected from the groupconsisting of D and N, X₂₃ is an amino acid selected from the groupconsisting of S and V, X₂₄ is an amino acid selected from the groupconsisting of A, S, and T, X₂₅ is an amino acid selected from the groupconsisting of A and I, X₂₆ is an amino acid selected from the groupconsisting of T and P, X₂₇ is an amino acid selected from the groupconsisting of S, N, and K, X₄₀ is any amino acid, X₄₂ is an amino acidselected from the group consisting of D and N, X₄₅ is any amino acid,X₄₉ is an amino acid selected from the group consisting of V and A, X₅₀is any amino acid, X₅₇ is any amino acid, and X₅₉ is any amino acid. Insome embodiments, peptides of the invention comprise or consist of asequence ofC-S-X₃-K-E-X₆-K-Q-X₉-T-X₁₁-X₁₂-X₁₃-X₁₄-G-L-K-Q-X₁₉-W-X₂₁-G-X₂₃-X₂₄-X₂₅-X₂₆-X₂₇-G-G-G-G-G-N-F-S-A-K-E-E-X₄₀-A-E-T-R-X₄₅-T-F-G-L-X₅₀-K-Q-Y-D-G-A-X₅₇-I-X₅₉-E-N-Q-V-Q-N-K-F-T-I-S-N-C(SEQ ID NO: 73), wherein X₃ is an amino acid selected from the groupconsisting of A and V, X₆ is an amino acid selected from the groupconsisting of E and D, X₉ is an amino acid selected from the groupconsisting of T and P, X₁₁ is an amino acid selected from the groupconsisting of T and V, X₁₂ is an amino acid selected from the groupconsisting of G and A, X₁₃ is an amino acid selected from the groupconsisting of L and V, X₁₄ is an amino acid selected from the groupconsisting of Y and F, X₁₉ is an amino acid selected from the groupconsisting of D and N, X₂₁ is an amino acid selected from the groupconsisting of D and N, X₂₃ is an amino acid selected from the groupconsisting of S and V, X₂₄ is an amino acid selected from the groupconsisting of A, S, and T, X₂₅ is an amino acid selected from the groupconsisting of A and I, X₂₆ is an amino acid selected from the groupconsisting of T and P, X₂₇ is an amino acid selected from the groupconsisting of S, N, and K, X₄₀ is any amino acid, X₄₅ is any amino acid,X₅₀ is any amino acid, X₅₇ is any amino acid, and X₅₉ is any amino acid.

The additional N-terminal peptide sequence can be a native sequence. Asused herein, a “native” sequence is a peptide sequence from anaturally-occurring Ehrlichia OMP-1 sequence, or a variant thereof. Incertain embodiments, the peptide sequence is a fragment of anaturally-occurring Ehrlichia OMP-1 sequence. The peptide sequence canbe, e.g., from a conserved or non-conserved region of OMP-l. The peptidesequence can comprise, e.g., an epitope, such as an immunodominantepitope or any other epitope recognizable by a host (e.g., human, dog,etc.) immune system. OMP-1 proteins and peptides thereof have beendescribed, e.g., in U.S. Pat. Nos. 6,544,517, 6,893,640, 6,923,963,7,063,846, and 7,407,770, U.S. Patent Applications 2004/0265333 and2009/0075368, and European Patent No. 1026949, the contents of which areincorporated herein by reference in their entirety.

Variant polypeptides are at least about 80, 85, 90, 95, 98, or 99%identical to a peptide shown in SEQ ID NOs: 1-73 and 83-86 and are alsopolypeptides of the invention. Percent sequence identity has an artrecognized meaning and there are a number of methods to measure identitybetween two polypeptide or polynucleotide sequences. See, e.g., Lesk,Ed., Computational Molecular Biology, Oxford University Press, New York,(1988); Smith, Ed., Biocomputing: Informatics And Genome Projects,Academic Press, New York, (1993); Griffin & Griffin, Eds., ComputerAnalysis Of Sequence Data, Part I, Humana Press, New Jersey, (1994); vonHeinje, Sequence Analysis In Molecular Biology, Academic Press, (1987);and Gribskov & Devereux, Eds., Sequence Analysis Primer, M StocktonPress, New York, (1991). Methods for aligning polynucleotides orpolypeptides are codified in computer programs, including the GCGprogram package (Devereux et al., Nuc. Acids Res. 12:387 (1984)),BLASTP, BLASTN, FASTA (Atschul et al., J Molec. Biol. 215:403 (1990)),and Bestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711) which uses the local homology algorithm ofSmith and Waterman (Adv. App. Math., 2:482-489 (1981)). For example, thecomputer program ALIGN which employs the FASTA algorithm can be used,with an affine gap search with a gap open penalty of −12 and a gapextension penalty of −2.

When using any of the sequence alignment programs to determine whether aparticular sequence is, for instance, about 95% identical to a referencesequence, the parameters are set such that the percentage of identity iscalculated over the full length of the reference polynucleotide and thatgaps in identity of up to 5% of the total number of nucleotides in thereference polynucleotide are allowed.

Variants of the peptide sequences can be readily selected by one ofskill in the art, based in part on known properties of the sequence. Forexample, a variant peptide can include amino acid substitutions (e.g.,conservative amino acid substitutions) and/or deletions (e.g., small,single amino acid deletions, or deletions encompassing 2, 3, 4, 5, 10,15, 20, or more contiguous amino acids). Thus, in certain embodiments, avariant of a native peptide sequence is one that differs from anaturally-occurring sequence by (i) one or more (e.g., 2, 3, 4, 5, 6, ormore) conservative amino acid substitutions, (ii) deletion of 1 or more(e.g., 2, 3, 4, 5, 6, or more) amino acids, or (iii) a combinationthereof. Deleted amino acids can be contiguous or non-contiguous.Conservative amino acid substitutions are those that take place within afamily of amino acids that are related in their side chains and chemicalproperties. These include, e.g., (1) acidic amino acids: aspartate,glutamate; (2) basic amino acids: lysine, arginine, histidine; (3)nonpolar amino acids: alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; (4) uncharged polar amino acids:glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine;(5) aliphatic amino acids: glycine, alanine, valine, leucine,isoleucine, serine, threonine, with serine and threonine optionallygrouped separately as aliphatic-hydroxyl; (6) aromatic amino acids:phenylalanine, tyrosine, tryptophan; (7) amide amino acids: asparagine,glutamine; and (9) sulfur-containing amino acids: cysteine andmethionine. See, e.g., Biochemistry, 2nd ed., Ed. by L. Stryer, W HFreeman and Co.: 1981. Methods for confirming that variant peptides aresuitable are conventional and routine.

Variants of the peptide sequences encompass variations on previouslydefined peptide sequences. For example, a previously described peptidesequence comprising a known epitope may be lengthened or shortened, atone or both ends (e.g., by about 1-3 amino acids), and/or one, two,three, four or more amino acids may be substituted by conservative aminoacids, etc. Furthermore, if a region of a protein has been identified ascontaining an epitope of interest, an investigator can “shift” theregion of interest (e.g., by about 5 amino acids in either direction)from the endpoints of the original rough region to optimize theactivity.

In certain embodiments, the additional N-terminal peptide sequence cancomprise or consist of another peptide having a sequence of SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO:83, or SEQ ID NO: 85. Thus, in some embodiments, a peptide of theinvention can be a multimer of sequences having a sequence of SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO:83, or SEQ ID NO: 85. In other embodiments, the N-terminal peptidesequence is a native OMP-1 peptide sequence that is naturally adjacentto the N-terminal end of a sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQID NO: 7, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 83, or SEQ ID NO: 85.In other embodiments, the peptide can comprise a fusion of sequences ofSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 70, SEQ ID NO: 72,SEQ ID NO: 83, or SEQ ID NO: 85 optionally through one or more linkingamino acids. For example, in one embodiment, the peptide can comprise asequence of SEQ ID NO: 1 linked to SEQ ID NO: 3 or SEQ ID NO: 85optionally through one or more linking amino acids (e.g. glycine,serine, or cysteine residues). In another embodiment, the peptide cancomprise a sequence of SEQ ID NO: 7 linked to SEQ ID NO: 3 or SEQ ID NO:85 optionally through one or more linking amino acids (e.g. glycine,serine, or cysteine residues). In another embodiment, the peptide cancomprise a sequence of SEQ ID NO: 83 linked to SEQ ID NO: 3 or SEQ IDNO: 85 optionally through one or more linking amino acids (e.g. glycine,serine, or cysteine residues). In still another embodiment, the peptidecan comprise a sequence of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 83linked to SEQ ID NO: 72 optionally through one or more linking aminoacids (e.g. glycine, serine, or cysteine residues). In yet anotherembodiment, the peptide can comprise a sequence of SEQ ID NO: 1, SEQ IDNO: 7, or SEQ ID NO: 83 linked to SEQ ID NO: 71 optionally through oneor more linking amino acids (e.g. glycine, serine, or cysteineresidues).

In certain embodiments, the additional N-terminal peptide sequence is anon-native sequence. As used herein, a “non-native” sequence is anyprotein sequence, whether from an Ehrlichia protein or otherwise, otherthan a native OMP-1 peptide sequence. In certain embodiments, theadditional N-terminal peptide sequence comprises an epitope of anEhrlichia surface antigen. In certain embodiments, the additionalN-terminal peptide sequence comprises an epitope of an Ehrlichiaantigen, such as p38, p43, p120, p140, p153, p156, p200, gp19, gp36,gp47, gp200, or HGE-3. Protein and peptide sequences corresponding toEhrlichia antigens have been described. See, e.g., U.S. Pat. Nos.6,306,402, 6,355,777, 7,204,992, and 7,407,770, and WO2006/138509, thecontents of which are incorporated herein by reference in theirentirety. Polypeptides or peptides derived from other microorganisms canalso be used.

In certain embodiments, the additional N-terminal peptide sequence is acombination of sequences. For example, the additional N-terminal peptidesequence can comprise a native sequence, a non-native sequence, or anycombination of such sequences (e.g., two or more native sequences, twoor more non-native sequence, or one or more native sequences incombination with one or more non-native sequences).

In certain embodiments, peptides of the invention comprise a sequencedefined by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 70, SEQID NO: 72, SEQ ID NO: 83, or SEQ ID NO: 85 and further comprise anadditional C-terminal sequence. The additional C-terminal peptidesequence can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,20, 25, or more amino acids. In certain embodiments, the additionalC-terminal sequence has a length of about 5 to about 10, about 10 toabout 15, about 15 to about 20, about 20 to about 25, about 25 to about30, about 30 to about 40, or about 40 to about 50 amino acids. Theadditional C-terminal peptide sequence can be a native OMP-1 sequence.In certain embodiments, the C-terminal peptide sequence is a fragment ofa naturally-occurring Ehrlichia OMP-1 sequence. The peptide sequence canbe, e.g., from a conserved or non-conserved region of OMP-1. The peptidesequence can comprise, e.g., an epitope, such as an immunodominantepitope or any other epitope recognizable by a host (e.g., human, dog,etc.) immune system. In certain embodiments, the additional C-terminalpeptide sequence can comprise or consist of another peptide having asequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 70, SEQID NO: 72, SEQ ID NO: 83, or SEQ ID NO: 85. For example, in certainembodiments, a peptide of the invention can be a multimer of sequenceseach having a sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7, SEQID NO: 70, SEQ ID NO: 72, SEQ ID NO: 83, or SEQ ID NO: 85. In otherembodiments, the native sequence is a OMP-1 sequence that is naturallyadjacent to the C-terminal end of a sequence of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 7, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 83, or SEQ IDNO: 85.

In certain embodiments, the additional C-terminal peptide sequence is anon-native sequence. In certain embodiments, the additional C-terminalpeptide sequence comprises an epitope of an Ehrlichia surface antigenother than OMP-1. In certain embodiments, the additional C-terminalpeptide sequence comprises an epitope of an Ehrlichia antigen, such asp38, p43, p120, p140, p153, p156, p200, gp19, gp36, gp47, gp200, orHGE-3. Polypeptides or peptides derived from other microorganisms canalso be used.

In certain embodiments, the additional C-terminal peptide sequence is acombination of sequences. For example, the additional C-terminal peptidesequence can comprise a native, a non-native sequence, or anycombination of such sequences (e.g., two or more native sequences, twoor more non-native sequence, or one or more native sequences incombination with one or more non-native sequences).

In certain embodiments, peptides of the invention comprise a sequencedefined by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 70, SEQID NO: 72, SEQ ID NO: 83, or SEQ ID NO: 85 and further comprise anadditional N-terminal peptide sequence and an additional C-terminalpeptide sequence. The additional N-terminal and C-terminal peptidesequences can be as described above. Peptides of the invention do notconsist of a full-length OMP-1 protein. However, in certain embodiments,peptides of the invention can comprise a full-length OMP-1 protein. Inother embodiments, peptides of the invention do not comprise afull-length OMP-1 protein.

A peptide of the invention comprising an additional N-terminal and/orC-terminal peptide sequence can be designed for diagnosing Ehrlichiainfections early after infection (e.g., within one to two weeks afterthe onset of infection). For example, in certain embodiments, theadditional N-terminal and/or C-terminal peptide sequence comprises anantigen or epitope associated with early stages of Ehrlichia infection.

In addition to the sequences described above, the additional N-terminaland C-terminal sequences can comprise or consist of a flexible sequence,designed to better present the peptides of the invention for detectionin an immunoassay (e.g., ELISA assay, lateral flow immunoassay,agglutination assay, etc.). Such flexible sequences can be readilyidentified by persons skilled in the art.

In certain embodiments, peptides of the invention comprise or consist of25 or more (e.g., 26, 27, 28, 29, or more) amino acid residues. Incertain embodiments, peptides of the invention comprise or consist of 30or more (e.g., 31, 32, 33, 34, or more) amino acid residues. In certainembodiments, peptides of the invention comprise or consist of 35 or more(e.g., 36, 37, 38, 39, or more) amino acid residues. In certainembodiments, peptides of the invention comprise or consist of 40 or more(e.g., 41, 42, 43, 44, or more) amino acid residues. In certainembodiments, peptides of the invention comprise or consist of 45 or more(e.g., 46, 47, 48, 49, or more) amino acid residues. In certainembodiments, peptides of the invention comprise or consist of 50 or more(e.g., 51, 52, 53, 54, or more) amino acid residues. In certainembodiments, peptides of the invention comprise or consist of 55, 60,65, 70, 75, 80, 85, 90, 95, 100, or more amino acid residues.

In certain embodiments, peptides of the invention comprise an epitope ofa peptide sequence described herein. For example, in certainembodiments, peptides of the invention comprise an epitope of a sequenceselected from the group consisting of SEQ ID NOs: 1-73 and 83-86.

In certain embodiments, peptides of the invention comprise a fragment ofa peptide sequence described herein. For example, in certainembodiments, peptides of the invention comprise a fragment of a sequenceselected from the group consisting of SEQ ID NOs: 1-73 and 83-86. Thefragment can be, e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44 amino acids in length. Thefragment can be contiguous or can include one or more deletions (e.g., adeletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid residues).For instance, in one embodiment, peptides of the invention comprise afragment of SEQ ID NO: 1. Such fragments may comprise at least 10, 15,20, 25, 30, or 35 contiguous amino acids from SEQ ID NO: 1. In someembodiments, the fragments comprise amino acids 1 to 26 of SEQ ID NO: 1.Thus, in one embodiment, a peptide of the invention comprises orconsists of a sequence ofS-X₂-K-E-D-K-Q-T-T-X₁₀-X₁₁-I-W-G-L-K-Q-X₁₈-W-X₂₀-G-X₂₂-P-X₂₄-X₂₅-X₂₆(SEQ ID NO: 84), wherein X₂ is an amino acid selected from the groupconsisting of A and V, X₁₀ is an amino acid selected from the groupconsisting of T and V, X₁₁ is an amino acid selected from the groupconsisting of G and A, X₁₈ is an amino acid selected from the groupconsisting of E and Q, X₂₀ is an amino acid selected from the groupconsisting of D and N, X₂₂ is an amino acid selected from the groupconsisting of S and V, X₂₄ is an amino acid selected from the groupconsisting of A and I, X₂₅ is an amino acid selected from the groupconsisting of T and P, and X₂₆ is an amino acid selected from the groupconsisting of S, N, and K.

In one particular embodiment, peptides of the invention comprise afragment of SEQ ID NO: 3 or SEQ ID NO: 72. Such fragments may compriseat least 10, 15, 20, 25, 30, 35, or 40 contiguous amino acids from SEQID NO: 3 or SEQ ID NO: 72. For instance, in certain embodiments, suchfragments may comprise amino acids 1 to 26 of SEQ ID NO: 3 or SEQ ID NO:72. Thus, in one embodiment, a peptide of the invention comprises orconsists of a sequence ofS-X₂-K-E-X₅-K-Q-X₈-T-X₁₀-X₁₁-X₁₂-X₁₃-G-L-K-Q-X₁₈-W-X₂₀-G-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆(SEQ ID NO: 86), wherein X₂ is an amino acid selected from the groupconsisting of A and V, X₅ is an amino acid selected from the groupconsisting of E and D, X₈ is an amino acid selected from the groupconsisting of T and P, X₁₀ is an amino acid selected from the groupconsisting of T and V, X₁₁ is an amino acid selected from the groupconsisting of G and A, X₁₂ is an amino acid selected from the groupconsisting of L and V, X₁₃ is an amino acid selected from the groupconsisting of Y and F, X₁₈ is an amino acid selected from the groupconsisting of D and N, X₂₀ is an amino acid selected from the groupconsisting of D and N, X₂₂ is an amino acid selected from the groupconsisting of S and V, X₂₃ is an amino acid selected from the groupconsisting of A, S, and T, X₂₄ is an amino acid selected from the groupconsisting of A and I, X₂₅ is an amino acid selected from the groupconsisting of T and P, and X₂₆ is an amino acid selected from the groupconsisting of S, N, and K. In other embodiments, the fragments maycomprise amino acids 33 to 71 of SEQ ID NO: 3 or SEQ ID NO: 72. Thus, inone embodiment, a peptide of the invention comprises or consists of asequence ofF-S-A-K-E-E-X₇-A-X₉-T-R-X₁₂-T-F-G-X₁₆-X₁₇-K-Q-Y-D-G-A-X₂₄-I-X₂₆-E-N-Q-V-Q-N-K-F-T-I-S-N-C(SEQ ID NO: 5), wherein X₇ is any amino acid, X₉ is an amino acidselected from the group consisting of D and N, X₁₂ is any amino acid,X₁₆ is an amino acid selected from the group consisting of V and A, X₁₇is any amino acid, X₂₄ is any amino acid, and X₂₆ is any amino acid. Inanother embodiment, a peptide of the invention comprises a sequence ofC-F-S-A-K-E-E-X₈-A-X₁₀-T-R-X₁₃-T-F-G-X₁₇-X₁₈-K-Q-Y-D-G-A-X₂₅-I-X₂₇-E-N-Q-V-Q-N-K-F-T-I-S-N(SEQ ID NO: 6), wherein X₈ is any amino acid, X₁₀ is an amino acidselected from the group consisting of D and N, X₁₃ is any amino acid,X₁₇ is an amino acid selected from the group consisting of V and A, X₁₈is any amino acid, X₂₅ is any amino acid, and X₂₇ is any amino acid. Instill another embodiment, a peptide of the invention comprises asequence ofF-S-A-K-E-E-X₇-A-E-T-R-X₁₂-T-F-G-L-X₁₇-K-Q-Y-D-G-A-X₂₄-I-X₂₆-E-N-Q-V-Q-N-K-F-T-I-S-N-C(SEQ ID NO: 71), wherein X₇ is any amino acid, X₁₂ is any amino acid,X₁₇ is any amino acid, X₂₄ is any amino acid, and X₂₆ is any amino acid.

In certain embodiments, the fragment comprises a sequence set forth inU.S. Pat. Nos. 6,306,402, 6,355,777, 7,204,992, or 7,407,770, or inWO2006/138509. In certain embodiments, the fragment does not consist ofa sequence set forth in one or more of U.S. Pat. Nos. 6,306,402,6,355,777, 7,204,992, and 7,407,770, and WO2006/138509. Peptides of theinvention that comprise a fragment of a peptide sequence describedherein can further comprise an additional N-terminal peptide sequence,an additional C-terminal peptide sequence, or a combination thereof. Theadditional N-terminal and C-terminal peptide sequences can be asdescribed above.

Peptides of the invention comprising an additional N-terminal orC-terminal peptide sequence can further comprise a linker connecting thepeptide (e.g., a peptide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7,SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 83, SEQ ID NO: 85, or afragment thereof) with the additional N-terminal or C-terminal peptidesequence. The linker can be, e.g., a peptide spacer. Such spacer canconsist of, for example, between about one and five (e.g., about three)amino acid residues, preferably uncharged amino acids, e.g., aliphaticresidues such as glycine or alanine. In one embodiment, the spacer is atriplet glycine spacer. In another embodiment, the spacer is a tripletalanine spacer. In yet another embodiment, the spacer comprises bothglycine and alanine residues. Alternatively, the linker can be achemical (i.e., non-peptide) linker.

In certain embodiments, peptides of the invention are produced bysynthetic chemistry (i.e., a “synthetic peptide”). In other embodiments,peptides of the invention are produced biologically (i.e., by cellularmachinery, such as a ribosome). In certain embodiments, peptides of theinvention are isolated. As used herein, an “isolated” peptide is apeptide that has been produced either synthetically or biologically andthen purified, at least partially, from the chemicals and/or cellularmachinery used to produce the peptide. In certain embodiments, anisolated peptide of the invention is substantially purified. The term“substantially purified,” as used herein, refers to a molecule, such asa peptide, that is substantially free of cellular material (proteins,lipids, carbohydrates, nucleic acids, etc.), culture medium, chemicalprecursors, chemicals used in synthesis of the peptide, or combinationsthereof. A peptide that is substantially purified has less than about40%, 30%, 25%, 20%, 15%, 10%, 5%, 2%, 1% or less of the cellularmaterial, culture medium, other polypeptides, chemical precursors,and/or chemicals used in synthesis of the peptide. Accordingly, asubstantially pure molecule, such as a peptide, can be at least about60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, by dry weight, themolecule of interest. An isolated peptide of the invention can be inwater, a buffer, or in a dry form awaiting reconstitution, e.g., as partof a kit. An isolated peptide of the present invention can be in theform of a pharmaceutically acceptable salt. Suitable acids and basesthat are capable of forming salts with the peptides of the presentinvention are well known to those of skill in the art, and includeinorganic and organic acids and bases.

In certain embodiments, peptides of the invention are affinity purified.For example, in certain embodiments, the peptides of the invention arepurified by means of their ability to bind to anti-Ehrlichia antibodies(e.g., antibodies to OMP-1 proteins and, optionally, other Ehrlichiaantigens) by contacting such antibodies with the peptides of theinvention such that peptide-antibody complexes are able to form, washingthe peptide-antibody complexes to remove impurities, and then elutingthe peptides from the antibodies. The antibodies can be, e.g., attachedto a solid support. Methods of affinity purification are well-known androutine to those skilled in the art.

In certain embodiments, peptides of the invention are modified. Thepeptides of the invention may be modified by a variety of techniques,such as by denaturation with heat and/or a detergent (e.g., SDS).Alternatively, peptides of the invention may be modified by associationwith one or more further moieties. The association can be covalent ornon-covalent, and can be, for example, via a terminal amino acid linker,such as lysine or cysteine, a chemical coupling agent, or a peptidebond. The additional moiety can be, for example, a ligand, a ligandreceptor, a fusion partner, a detectable label, an enzyme, or asubstrate that immobilizes the peptide.

Peptides of the invention can be conjugated to a ligand, such as biotin(e.g., via a cysteine or lysine residue), a lipid molecule (e.g., via acysteine residue), or a carrier protein (e.g., serum albumin,immunoglobulin Fc domain, keyhole limpet hemocyanin (KLH) via e.g., acysteine or lysine residue). Attachment to ligands, such as biotin, canbe useful for associating the peptide with ligand receptors, such asavidin, streptavidin, polymeric streptavidin (see e.g., US 2010/0081125and US 2010/0267166, both of which are herein incorporated byreference), or neutravidin. Avidin, streptavidin, polymericstreptavidin, or neutravidin, in turn, can be linked to a signalingmoiety (e.g., an enzyme, such as horse radish peroxidase (HRP) oralkaline phosphatase (ALP), or other moiety that can be visualized, suchas a metallic nanoparticle or nanoshell (e.g., colloidal gold) or afluorescent moiety) or a solid substrate (e.g., an Immobilon™ ornitrocellulose membrane). Alternatively, the peptides of the inventioncan be fused or linked to a ligand receptor, such as avidin,streptavidin, polymeric streptavidin, or neutravidin, therebyfacilitating the association of the peptides with the correspondingligand, such as biotin and any moiety (e.g., signaling moiety) or solidsubstrate attached thereto. Examples of other ligand-receptor pairs arewell-known in the art and can similarly be used.

Peptides of the invention can be fused to a fusion partner (e.g., apeptide or other moiety) that can be used to improve purification, toenhance expression of the peptide in a host cell, to aid in detection,to stabilize the peptide, etc. Examples of suitable compounds for fusionpartners include carrier proteins (e.g., serum albumin, immunoglobulinFc domain, KLH), enzymes (e.g., horse radish peroxidase (HRP),beta-galactosidase, glutathione-S-transferase, alkaline phosphatase), ahistidine tag, etc. The fusion can be achieved by means of, e.g., apeptide bond. For example, peptides of the invention and fusion partnerscan be fusion proteins and can be directly fused in-frame or cancomprise a peptide linker, as discussed above in the context ofadditional N-terminal and C-terminal peptide sequences. In certainembodiments, a mixture of peptides of the invention can be linked by adendrimer, e.g., as in a MAPS structure.

In addition, peptides of the invention may be modified to include any ofa variety of known chemical groups or molecules. Such modificationsinclude, but are not limited to, glycosylation, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment to polyethylene glycol(e.g., PEGylation), covalent attachment of flavin, covalent attachmentof a heme moiety, covalent attachment of a nucleotide or nucleotidederivative, covalent attachment of a lipid or lipid derivative, covalentattachment of phosphatidylinositol, cross-linking, cyclization,disulfide bond formation, demethylation, formation of covalentcross-links, formation of cystine, formation of pyroglutamate,formylation, gamma carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, ubiquitination, modifications with fattyacids, transfer-RNA mediated addition of amino acids to proteins such asarginylation, etc. Analogues of an amino acid (including unnatural aminoacids) and peptides with substituted linkages are also included.Peptides of the invention that consist of any of the sequences discussedherein may be modified by any of the discussed modifications. Suchpeptides still “consist of” the amino acids.

Modifications as set forth above are well-known to those of skill in theart and have been described in great detail in the scientificliterature. Several particularly common modifications, glycosylation,lipid attachment, sulfation, gamma-carboxylation of glutamic acidresidues, hydroxylation and ADP-ribosylation, for instance, aredescribed in many basic texts, such as Proteins-Structure and MolecularProperties, 2nd ed., T. E. Creighton, W.H. Freeman and Company, New York(1993). Many detailed reviews are available on this subject, such as byWold, F., Posttranslational Covalent Modification of Proteins, B. C.Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al.(1990) Meth. Enzymol. 182:626-646 and Rattan et al. (1992) Ann. N.Y.Acad. Sci. 663:48-62.

In certain embodiments, peptides of the invention are attached to orimmobilized on a substrate, such as a solid or semi-solid support. Theattachment can be covalent or non-covalent, and can be facilitated by amoiety associated with the peptide that enables covalent or non-covalentbinding, such as a moiety that has a high affinity to a componentattached to the carrier, support or surface. For example, the peptidecan be associated with a ligand, such as biotin, and the componentassociated with the surface can be a corresponding ligand receptor, suchas avidin. In some embodiments, the peptide can be associated with afusion partner, e.g., bovine serum albumin (BSA), which facilitates theattachment of the peptide to a substrate. In other embodiments, thepeptides of the invention are attached to or immobilized on a substratevia a metallic nanolayer. In one embodiment, the metallic nanolayer iscomprised of cadmium, zinc, mercury, or a noble metal, such as gold,silver, copper, and platinum. The peptide or mixture of peptides can beattached to or immobilized on the substrate either prior to or after theaddition of a sample containing antibody during an immunoassay.

In certain embodiments, the substrate is a bead, such as a colloidalparticle (e.g., a colloidal nanoparticle made from gold, silver,platinum, copper, cadmium, metal composites, other soft metals,core-shell structure particles, or hollow gold nanospheres) or othertype of particle (e.g., a magnetic bead or a particle or nanoparticlecomprising silica, latex, polystyrene, polycarbonate, polyacrylate, orPVDF). Such particles can comprise a label (e.g., a colorimetric,chemiluminescent, or fluorescent label) and can be useful forvisualizing the location of the peptides during immunoassays. In certainembodiments, a terminal cysteine of a peptide of the invention is usedto bind the peptide directly to the nanoparticles made from gold,silver, platinum, copper, cadmium, metal composites, or other softmetals, or metallic nanoshells (e.g., gold hollow spheres, gold-coatedsilica nanoshells, and silica-coated gold shells).

In certain embodiments, the substrate is a dot blot or a flow path in alateral flow immunoassay device. For example, the peptides can beattached or immobilized on a porous membrane, such as a PVDF membrane(e.g., an Immobilon™ membrane), a nitrocellulose membrane, polyethylenemembrane, nylon membrane, or a similar type of membrane.

In certain embodiments, the substrate is a flow path in an analytical orcentrifugal rotor. In other embodiments, the substrate is a tube or awell, such as a well in a plate (e.g., a microtiter plate) suitable foruse in an ELISA assay. Such substrates can comprise glass,cellulose-based materials, thermoplastic polymers, such as polyethylene,polypropylene, or polyester, sintered structures composed of particulatematerials (e.g., glass or various thermoplastic polymers), or castmembrane film composed of nitrocellulose, nylon, polysulfone, or thelike. A substrate can be sintered, fine particles of polyethylene,commonly known as porous polyethylene, for example, 0.2-15 micron porouspolyethylene from Chromex Corporation (Albuquerque, N. Mex.). All ofthese substrate materials can be used in suitable shapes, such as films,sheets, or plates, or they may be coated onto or bonded or laminated toappropriate inert carriers, such as paper, glass, plastic films, orfabrics. Suitable methods for immobilizing peptides on solid phasesinclude ionic, hydrophobic, covalent interactions and the like.

Accordingly, in another aspect, the invention provides devices. Incertain embodiments, the devices are useful for performing animmunoassay. For example, in certain embodiments, the device is alateral flow immunoassay device. In some embodiments, the device is aslide comprised of a plurality of beads to which a peptide or populationof peptides is attached. In other embodiments, the device is ananalytical or centrifugal rotor. In other embodiments, the device is adot blot, slot blot, or Western blot. In other embodiments, the deviceis a tube or a well, e.g., in a plate suitable for an ELISA assay. Instill other embodiments, the device is an electrochemical sensor, anoptical sensor, or an opto-electronic sensor.

In certain embodiments, the device comprises a peptide or population ofpeptides of the invention. In other embodiments, the device comprises amixture of different peptides of the invention. For example, in certainembodiments, the device comprises two, three, four, or more differentpeptides of the invention. In certain embodiments, the peptide or eachpeptide in the mixture comprises a sequence of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 7, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 83, SEQ ID NO:85 or a fragment thereof. In other embodiments, the peptide or eachpeptide in the mixture comprises a sequence of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 72, or a fragment thereof. In certain embodiments, themixture or population of peptides are attached to or immobilized uponthe device optionally through a metallic nanolayer. The devices may beused to detect the presence of antibodies to Ehrlichia antigens frommultiple species (e.g., E. canis, E. chaffeensis, E. ewingii, and E.muris) in a sample simultaneously. In one embodiment, the devicecomprises a population of isolated peptides comprising three or moredifferent peptides, wherein each peptide in the population comprises asequence of SEQ ID NO: 72. In related embodiments, the population ofisolated peptides further comprises peptides comprising a sequence ofSEQ ID NO: 3 and/or SEQ ID NO: 71. In another embodiment, the devicecomprises a population of isolated peptides comprising three or moredifferent peptides, wherein each peptide in the population comprises asequence of SEQ ID NO: 3. In another embodiment, the device comprises apopulation of isolated peptides comprising three or more differentpeptides, wherein each peptide in the population comprises a sequence ofSEQ ID NO: 1. In still another embodiment, the device comprises apopulation of isolated peptides comprising three or more differentpeptides, wherein each peptide in the population comprises a sequence ofSEQ ID NO: 71. In other embodiments, the device comprises a populationof isolated peptides comprising three or more different peptides,wherein each peptide in the population comprises a sequence of SEQ IDNO: 7, SEQ ID NO: 70, SEQ ID NO: 83, or SEQ ID NO: 85.

In another aspect, the invention provides compositions comprising one ormore peptides of the invention. For example, in certain embodiments, theinvention provides a composition comprising a peptide comprising asequence of SEQ ID NO: 1, or mixtures thereof. In certain embodiments,the composition comprises a mixture of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500,or more peptides (e.g., all possible peptides defined by SEQ ID NO: 1).Thus, the present invention provides a population of isolated peptidescomprising three or more different peptides, wherein each peptide in thepopulation comprises a sequence of SEQ ID NO: 1. In certain embodiments,the peptides in the population or mixture comprise an N-terminal and/orC-terminal addition, and/or are modified (e.g., by association with oneor more further moieties), as described herein. In certain embodiments,the peptides comprise the same N-terminal and/or C-terminal additions.In other embodiments, the peptides comprise different N-terminal and/orC-terminal additions.

In still other embodiments, the invention provides a compositioncomprising a peptide comprising a sequence of SEQ ID NO: 3, SEQ ID NO:5, SEQ ID NO: 7, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 83, SEQ ID NO:85, or mixtures thereof. In certain embodiments, the compositioncomprises a mixture of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40,50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, or more peptides(e.g., all possible peptides defined by SEQ ID NO: 3, SEQ ID NO: 5, SEQID NO: 7, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 83, or SEQ ID NO:85). Thus, the invention provides a population of isolated peptidescomprising three or more different peptides, wherein each peptide in thepopulation comprises a sequence of SEQ ID NO: 3. In another embodiment,the invention provides a population of isolated peptides comprisingthree or more different peptides, wherein each peptide in the populationcomprises a sequence of SEQ ID NO: 72. In other embodiments, theinvention provides a population of isolated peptides comprising three ormore different peptides, wherein each peptide in the populationcomprises a sequence of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 70, SEQID NO: 83, or SEQ ID NO: 85. The peptides in the population or mixturemay comprise an N-terminal and/or C-terminal addition, and/or bemodified (e.g., by association with one or more further moieties), asdescribed herein.

In certain embodiments, the compositions comprise one or more peptides(or one or more populations of peptides) of the invention and one ormore additional peptides, such as an Ehrlichia peptide or antigen, apeptide or antigen from one or more infectious Ehrlichia species, or apeptide or antigen from one or more causative agents of monocytic and/orgranulocytic ehrlichiosis. The Ehrlichia peptide or antigen can be anyEhrlichia surface peptide or antigen, or any peptide or antigendescribed herein (e.g., any peptide or antigen of an OMP-1, p38, p43,p120, p140, p153, p156, p200, gp19, gp36, gp47, gp200, or HGE-3 protein,or any fragment or epitope thereof). The combination may comprise acocktail (a simple mixture) of individual peptides or polypeptides, itmay be in the form of a fusion peptide or polypeptide (e.g., amultimeric peptide), or the peptides may be linked by a dendrimer (e.g.,as in a MAPS structure) optionally through a linking residue (e.g.lysine or cysteine residue). For instance, in certain embodiments, acomposition comprises one or more peptides of the invention (e.g., apeptide having a sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 83, or SEQ ID NO:85) and one or more antigenic Ehrlichia peptides having a sequence ofF-S-A-K-E-E-X₇-A-E-T-R-X₁₂-T-F-G-L-X₁₇-K-Q-Y-D-G-A-X₂₄-I-X₂₆-E-N-Q-V-Q-N-K-F-T-I-S-N-C(SEQ ID NO: 71) or a fragment thereof, wherein X₇ is any amino acid, X₁₂is any amino acid, X₁₇ is any amino acid, X₂₄ is any amino acid, and X₂₆is any amino acid. In some embodiments, X₇ is K. In other embodiments,X₁₂ is an amino acid selected from the group consisting of K and R, andX₁₇ is an amino acid selected from the group consisting of E and D. Instill other embodiments, X₂₄ is an amino acid selected from the groupconsisting of K and Q, and X₂₆ is an amino acid selected from the groupconsisting of E and T.

In certain embodiments, the mixture or population of peptides comprisesone or more peptides having a sequence of SEQ ID NO: 3 and one or morepeptides having a sequence of SEQ ID NO: 71. In still other embodiments,the mixture or population of peptides comprises one or more peptideshaving a sequence of SEQ ID NO: 1 and one or more peptides having asequence of SEQ ID NO: 71. In one embodiment, the mixture or populationof peptides comprises one or more peptides having a sequence of SEQ IDNO: 3 and one or more peptides having a sequence of SEQ ID NO: 72. Inanother embodiment, the mixture or population of peptides comprises oneor more peptides having a sequence of SEQ ID NO: 72 and one or morepeptides having a sequence of SEQ ID NO: 71. In one particularembodiment, the mixture or population of peptides comprises one or morepeptides having a sequence of SEQ ID NO: 3, one or more peptides havinga sequence of SEQ ID NO: 72, and one or more peptides having a sequenceof SEQ ID NO: 71. Such mixtures enable the detection of antibodies toEhrlichia antigens from multiple species (e.g., E. canis, E.chaffeensis, E. ewingii, and E. muris) in a sample simultaneously.

A peptide of the invention may be fused at its N-terminus or C-terminusto another suitable peptide. Two or more copies of a peptide of theinvention may be joined to one another, alone or in combination with oneor more additional peptides. Combinations of fused and unfused peptidesor polypeptides can be used. In one embodiment, the additionalpeptide(s) contain B-cell and/or T-cell epitopes from an Ehrlichiapeptide or antigen, a peptide or antigen from an infectious Ehrlichiaspecies, or a peptide or antigen from a causative agent of monocyticand/or granulocytic ehrlichiosis.

In another aspect, the invention provides nucleic acids comprising asequence encoding a peptide of the invention. Nucleic acids of theinvention contain less than an entire microbial genome and can besingle- or double-stranded. A nucleic acid can be RNA, DNA, cDNA,genomic DNA, chemically synthesized RNA or DNA or combinations thereof.The nucleic acids can be purified free of other components, such asproteins, lipids and other polynucleotides. For example, the nucleicacids can be 50%, 75%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% purified.The nucleic acids of the invention encode the peptides described herein.In certain embodiments, the nucleic acids encode a peptide having thesequence of SEQ ID NOs: 1-73 and 83-86, or combinations thereof. Nucleicacids of the invention can comprise other nucleotide sequences, such assequences coding for linkers, signal sequences, TMR stop transfersequences, transmembrane domains, or ligands useful in proteinpurification such as glutathione-S-transferase, histidine tag, andstaphylococcal protein A.

Nucleic acids of the invention can be isolated. An “isolated” nucleicacids is one that is not immediately contiguous with one or both of the5′ and 3′ flanking genomic sequences with which it is naturallyassociated. An isolated nucleic acid can be, e.g., a recombinant DNAmolecule of any length, provided that the nucleic acid sequencesnaturally found immediately flanking the recombinant DNA molecule in anaturally-occurring genome is removed or absent. Isolated nucleic acidsalso include non-naturally occurring nucleic acid molecules. Nucleicacids of the invention can also comprise fragments that encodeimmunogenic peptides. Nucleic acids of the invention can encodefull-length polypeptides, peptide fragments, and variant or fusionpeptides.

Nucleic acids of the invention can be isolated, at least in part, fromnucleic acid sequences present in, for example, a biological sample,such as blood, serum, saliva, or tissue from an infected individual.Nucleic acids can also be synthesized in the laboratory, for example,using an automatic synthesizer. An amplification method such as PCR canbe used to amplify nucleic acids, at least in part, from either genomicDNA or cDNA encoding the polypeptides.

Nucleic acids of the invention can comprise coding sequences fornaturally occurring polypeptides or can encode altered sequences that donot occur in nature. If desired, nucleic acids can be cloned into anexpression vector comprising expression control elements, including forexample, origins of replication, promoters, enhancers, or otherregulatory elements that drive expression of the polynucleotides of theinvention in host cells. An expression vector can be, for example, aplasmid, such as pBR322, pUC, or ColE1, or an adenovirus vector, such asan adenovirus Type 2 vector or Type 5 vector. Optionally, other vectorscan be used, including but not limited to Sindbis virus, simian virus40, alphavirus vectors, poxvirus vectors, and cytomegalovirus andretroviral vectors, such as murine sarcoma virus, mouse mammary tumorvirus, Moloney murine leukemia virus, and Rous sarcoma virus.Minichromosomes such as MC and MC1, bacteriophages, phagemids, yeastartificial chromosomes, bacterial artificial chromosomes, virusparticles, virus-like particles, cosmids (plasmids into which phagelambda cos sites have been inserted) and replicons (genetic elementsthat are capable of replication under their own control in a cell) canalso be used.

Methods for preparing polynucleotides operably linked to an expressioncontrol sequence and expressing them in a host cell are well-known inthe art. See, e.g., U.S. Pat. No. 4,366,246. A nucleic acid of theinvention is operably linked when it is positioned adjacent to or closeto one or more expression control elements, which direct transcriptionand/or translation of the polynucleotide.

Thus, for example, a peptide of the invention can be producedrecombinantly following conventional genetic engineering techniques. Toproduce a recombinant peptide of the invention, a nucleic acid encodingthe peptide is inserted into a suitable expression system. Generally, arecombinant molecule or vector is constructed in which thepolynucleotide sequence encoding the selected peptide is operably linkedto an expression control sequence permitting expression of the peptide.Numerous types of appropriate expression vectors are known in the art,including, e.g., vectors containing bacterial, viral, yeast, fungal,insect or mammalian expression systems. Methods for obtaining and usingsuch expression vectors are well-known. For guidance in this and othermolecular biology techniques used for compositions or methods of theinvention, see, e.g., Sambrook et al., Molecular Cloning, A LaboratoryManual, current edition, Cold Spring Harbor Laboratory, New York; Milleret al, Genetic Engineering, 8:277-298 (Plenum Press, current edition),Wu et al., Methods in Gene Biotechnology (CRC Press, New York, N.Y.,current edition), Recombinant Gene Expression Protocols, in Methods inMolecular Biology, Vol. 62, (Tuan, ed., Humana Press, Totowa, N.J.,current edition), and Current Protocols in Molecular Biology, (Ausabelet al., Eds.,) John Wiley & Sons, NY (current edition), and referencescited therein.

Accordingly, the invention also provides vectors comprising nucleicacids of the invention, and host cells comprising such vectors. Incertain embodiments, the vector is a shuttle vector. In otherembodiments, the vector is an expression vector (e.g., a bacterial oreukaryotic expression vector). In certain embodiments, the host cell isa bacterial cell. In other embodiments, the host cell is a eukaryoticcell.

Suitable host cells or cell lines for the recombinant nucleic acids orvectors of the invention transfection by this method include bacterialcells. For example, various strains of E. coli (e.g., HB101, MC1061) arewell-known as host cells in the field of biotechnology. Various strainsof B. subtilis, Pseudomonas, Streptomyces, and other bacilli and thelike can also be employed in this method. Alternatively, a peptide ofthe invention can be expressed in yeast, insect, mammalian, or othercell types, using conventional procedures. Cell-free in vitro synthesisand/or enzyme-mediated synthetic machineries may also be used.

The present invention also provides a method for producing a recombinantpeptide or polypeptide, which involves transfecting or transforming,e.g., by conventional means such as electroporation, a host cell with atleast one expression vector containing a polynucleotide of the inventionunder the control of an expression control sequence (e.g., atranscriptional regulatory sequence). The transfected or transformedhost cell is then cultured under conditions that allow expression of thepeptide or polypeptide. The expressed peptide or polypeptide isrecovered, isolated, and optionally purified from the cell (or from theculture medium, if expressed extracellularly) by appropriate means knownto one of skill in the art, including liquid chromatography such asnormal or reversed phase, using HPLC, FPLC and the like, affinitychromatography, such as with inorganic ligands or monoclonal antibodies,size exclusion chromatography, immobilized metal chelate chromatography,gel electrophoresis, and the like. One of skill in the art may selectthe most appropriate isolation and purification techniques withoutdeparting from the scope of this invention. One skilled in the art candetermine the purity of the peptide or polypeptide by using standardmethods including, e.g., polyacrylamide gel electrophoresis (e.g.,SDS-PAGE), capillary electrophoresis, column chromatography (e.g., highperformance liquid chromatography (HPLC)), amino-terminal amino acidanalysis, and quantitative amino acid analysis.

Methods

In another aspect, the invention provides methods of detecting in asample an antibody to an epitope of an Ehrlichia antigen. In certainembodiments, the methods comprise contacting a sample with a peptide ofthe invention, and detecting formation of an antibody-peptide complexcomprising said peptide, wherein formation of said complex is indicativeof the presence of an antibody to an epitope of an Ehrlichia antigen insaid sample. In some embodiments, the Ehrlichia antigen is from aninfectious Ehrlichia species. In certain embodiments, the Ehrlichiaantigen is from a pathogenic Ehrlichia species, such as Ehrlichiachaffeensis, E. ewingii, E. muris, or Ehrlichia canis. Other species ofEhrlichia which have been implicated in monocytic and/or granulocyticehrlichiosis can also be detected using the methods of the invention,provided they induce antibodies which can react specifically with apeptide of the invention. Thus, it is to be understood that the term“pathogenic Ehrlichia,” as used herein, refers to any such Ehrlichiaspecies that causes monocytic and/or granulocytic ehrlichiosis. Inparticular embodiments, the methods provide detection of antibodies toEhrlichia antigens from multiple species (e.g., E. canis, E.chaffeensis, E. ewingii, and E. muris) in a sample simultaneously.

In certain embodiments, the methods comprise contacting the sample witha mixture or population of two, three, four, or more (e.g., 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300,400, 500, or more) different peptides of the invention. For instance, inone particular embodiment, the methods comprise contacting the samplewith a mixture or population of two or more different isolated peptides,wherein each isolated peptide comprises a sequence of SEQ ID NO: 1. Inanother particular embodiment, the methods comprise contacting thesample with a mixture or population of two or more different isolatedpeptides, wherein each isolated peptide comprises a sequence of SEQ IDNO: 3. In still another embodiment, the methods comprise contacting thesample with a mixture or population of two or more different isolatedpeptides, wherein each isolated peptide comprises a sequence of SEQ IDNO: 72. In some embodiments, the methods comprise contacting the samplewith a mixture or population of two or more different isolated peptides,wherein each isolated peptide comprises a sequence of SEQ ID NO: 1, SEQID NO: 3, or SEQ ID NO: 72. In other embodiments, the methods comprisecontacting the sample with a mixture or population of two or moredifferent isolated peptides, wherein each isolated peptide comprises asequence of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 70, SEQ ID NO: 83, orSEQ ID NO: 85.

In certain embodiments, the methods comprise contacting the sample witha mixture of one or more peptides of the invention and one or more otherpeptides (e.g., an Ehrlichia peptide, or antigenic fragment or epitopethereof, such as an Ehrlichia surface antigen, or an OMP-1, p38, p43,p120, p140, p153, p156, p200, gp19, gp36, gp47, gp200, or HGE-3protein). For instance, in some embodiments, the methods comprisecontacting the sample with a mixture of one or more peptides of theinvention (e.g., SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 83, or SEQ ID NO: 85) and oneor more Ehrlichia antigenic peptides having a sequence of SEQ ID NO: 71.In one particular embodiment, the methods comprise contacting the samplewith a mixture of one or more peptides having a sequence of SEQ ID NO: 3and one or more peptides having a sequence of SEQ ID NO: 71. In anotherembodiment, the methods comprise contacting the sample with a mixture ofone or more peptides having a sequence of SEQ ID NO: 72 and one or morepeptides having a sequence of SEQ ID NO: 71. In another embodiment, themethods comprise contacting the sample with a mixture of one or morepeptides having a sequence of SEQ ID NO: 72 and one or more peptideshaving a sequence of SEQ ID NO: 3. In one embodiment, the methodscomprise contacting the sample with a mixture of one or more peptideshaving a sequence of SEQ ID NO: 72, one or more peptides having asequence of SEQ ID NO: 3, and one or more peptides having a sequence ofSEQ ID NO: 71. Mixtures of peptides of the invention allow, in someembodiments, for the detection of antibodies to antigens from multipleEhrlichia species (e.g., E. canis, E. muris, E. chaffeensis, and E.ewingii) in a sample simultaneously.

In certain embodiments, the peptide or each peptide in the mixture orpopulation is an isolated (e.g., synthetic and/or purified) peptide. Incertain embodiments, the peptide or mixture of peptides (i.e. populationof peptides) is attached to or immobilized upon a solid support. Incertain embodiments, the solid support is a bead (e.g., a metallicnanoparticle or nanoshell, a nanoparticle, a latex bead, etc.), a flowpath in a lateral flow immunoassay device (e.g., a porous membrane), aflow path in an analytical or centrifugal rotor, a blot (Western blot,dot blot, or slot blot), a tube or a well (e.g., in a plate suitable foran ELISA assay), or a sensor (e.g., an electrochemical, optical, oropto-electronic sensor). In some embodiments, the peptide or mixture ofpeptides is attached to or immobilized upon a solid support through ametallic nanolayer that, in some embodiments, may be comprised ofcadmium, zinc, mercury, or a noble metal (e.g., gold, silver, copper,and platinum).

In certain embodiments, the detecting step comprises performing an ELISAor immunofluorescence assay. In other embodiments, the detecting stepcomprises performing a lateral flow immunoassay. In other embodiments,the detecting step comprises performing an agglutination assay (e.g., ahemagglutination or particle/bead agglutination assay). In otherembodiments, the detecting step comprises spinning the sample in ananalytical or centrifugal rotor. In some embodiments, the detecting stepcomprises performing a wavelength shift assay. Such wavelength shiftassays may entail measuring or determining a change in the surfaceplasmon resonance or localized surface plasmon resonance wavelengthresulting from binding of antibodies to peptides attached to metallicnanolayers or metallic nanoparticle/nanoshells. In still otherembodiments, the detecting step comprises analyzing the sample with anelectrochemical, optical, or opto-electronic sensor.

There are a number of different conventional assays for detectingformation of an antibody-peptide complex comprising a peptide of theinvention. For example, the detecting step can comprise performing anELISA assay, performing an immunofluorescence assay, performing alateral flow immunoassay, performing an agglutination assay, performinga wavelength shift assay, performing a Western blot, slot blot, or dotblot, analyzing the sample in an analytical or centrifugal rotor, oranalyzing the sample with an electrochemical, optical, oropto-electronic sensor. These different assays are described hereinand/or are well-known to those skilled in the art.

In one embodiment, the methods involve detecting the presence ofnaturally occurring antibodies against one or more Ehrlichia antigens(e.g., the antigen of a pathogenic Ehrlichia, such as E. chaffeensis, E.muris, E. ewingii, or E. canis) which are produced by the infectedsubject's immune system in its biological fluids or tissues, and whichare capable of binding specifically to a peptide of the invention orcombinations of a peptide of the invention and, optionally, one or moresuitable additional antigenic polypeptides or peptides.

Suitable immunoassay methods typically include: receiving or obtaining(e.g., from a patient) a sample of body fluid or tissue likely tocontain antibodies; contacting (e.g., incubating or reacting) a sampleto be assayed with a peptide of the invention, under conditionseffective for the formation of a specific peptide-antibody complex(e.g., for specific binding of the peptide to the antibody); andassaying the contacted (reacted) sample for the presence of anantibody-peptide reaction (e.g., determining the amount of anantibody-peptide complex). The presence of an elevated amount of theantibody-peptide complex indicates that the subject was exposed to andinfected with an infectious Ehrlichia species. A peptide, including amodified form thereof, which “binds specifically” to (e.g., “is specificfor” or binds “preferentially” to) an antibody against an Ehrlichiaantigen interacts with the antibody, or forms or undergoes a physicalassociation with it, in an amount and for a sufficient time to allowdetection of the antibody. By “specifically” or “preferentially,” it ismeant that the peptide has a higher affinity (e.g., a higher degree ofselectivity) for such an antibody than for other antibodies in a sample.For example, the peptide can have an affinity for the antibody of atleast about 1.5-fold, 2-fold, 2.5-fold, 3-fold, or higher than for otherantibodies in the sample. Such affinity or degree of specificity can bedetermined by a variety of routine procedures, including, e.g.,competitive binding studies. In an ELISA assay, a positive response isdefined as a value 2 or 3 standard deviations greater than the meanvalue of a group of healthy controls. In some embodiments, a second tierassay is required to provide an unequivocal serodiagnosis of monocyticand/or granulocytic ehrlichiosis.

Phrases such as “sample containing an antibody” or “detecting anantibody in a sample” are not meant to exclude samples or determinations(e.g., detection attempts) where no antibody is contained or detected.In a general sense, this invention involves assays to determine whetheran antibody produced in response to infection with an infectiousEhrlichia is present in a sample, irrespective of whether or not it isdetected.

Conditions for reacting peptides and antibodies so that they reactspecifically are well-known to those of skill in the art. See, e.g.,Current Protocols in Immunology (Coligan et al., editors, John Wiley &Sons, Inc).

The methods comprise receiving or obtaining a sample of body fluid ortissue likely to contain antibodies from a subject. The antibodies canbe, e.g., of IgG, IgE, IgD, IgM, or IgA type. Generally, IgM and/or IgAantibodies are detected, e.g., for detection at early stages ofinfection. IgG antibodies can be detected when some of the additionalpeptides discussed above are used in the method (e.g., peptides for thedetection of flagellum proteins). The sample is preferably easy toobtain and may be whole blood, plasma, or serum derived from a venousblood sample or even from a finger prick. Tissue from other body partsor other bodily fluids, such as cerebro-spinal fluid (CSF), saliva,gastric secretions, mucus, urine, etc., are known to contain antibodiesand may be used as a source of the sample. The sample may also be atissue extract or a cell lysate.

Once the peptide antigen and sample antibody are permitted to react in asuitable medium, an assay is performed to determine the presence orabsence of an antibody-peptide reaction. Among the many types ofsuitable assays, which will be evident to a skilled worker, areimmunoprecipitation and agglutination assays.

In certain embodiments of the invention, the assay comprises:immobilizing the antibody(s) in the sample; adding a peptide of theinvention; and detecting the degree of antibody bound to the peptide,e.g., by the peptide being labeled or by adding a labeled substance,such as a labeled binding partner (e.g., streptavidin-HRP orstreptavidin-colloidal gold complex) or a labeled antibody whichspecifically recognizes the peptide. See, e.g., FIG. 2. In otherembodiments, the assay comprises: immobilizing a peptide of theinvention; adding the sample containing antibodies; and detecting theamount of antibody bound to the peptide, e.g., by adding another peptideof the invention conjugated, directly or indirectly, to a label (e.g.,metallic nanoparticle or metallic nanoshell, fluorescent label, enzyme(e.g., horseradish peroxidase or alkaline phosphatase)) or by adding alabeled substance, such as a binding partner or a labeled antibody whichspecifically recognizes the sample antibodies (e.g., anti-human IgGantibodies, anti-human IgM antibodies, anti-dog IgG antibodies, anti-dogIgM antibodies, anti-cat IgG antibodies, anti-cat IgM antibodies,protein A, protein G, protein A/G fusion proteins, protein L, orcombinations thereof, etc.). See, e.g., FIGS. 1, 3, and 4. In otherembodiments, the assay comprises: immobilizing a peptide of theinvention; adding the sample containing antibodies; and detecting theamount of antibody bound to the peptide, e.g., by adding a first bindingpartner which specifically recognizes the sample antibodies (e.g.,anti-human IgG antibodies, anti-human IgM antibodies, anti-dog IgGantibodies, anti-dog IgM antibodies, anti-cat IgG antibodies, anti-catIgM antibodies, protein A, protein G, protein A/G fusion proteins,protein L, etc.), and further adding a second binding partner (e.g.,protein A, protein G, protein A/G fusion proteins, protein L, etc.),wherein the second binding partner is labeled and recognizes said firstbinding partner. In still other embodiments, the assay comprises:reacting the peptide and the sample containing antibodies without any ofthe reactants being immobilized, and then detecting the amount ofcomplexes of antibody and peptide, e.g., by the peptide being labeled orby adding a labeled substance, such as a labeled binding partner (e.g.,streptavidin-HRP or streptavidin-colloidal gold complex) or a labeledantibody which specifically recognizes the peptide.

Immobilization of a peptide of the invention can be either covalent ornon-covalent, and the non-covalent immobilization can be non-specific(e.g., non-specific binding to a polystyrene surface in, e.g., amicrotiter well). Specific or semi-specific binding to a solid orsemi-solid carrier, support or surface, can be achieved by the peptidehaving, associated with it, a moiety which enables its covalent ornon-covalent binding to the solid or semi-solid carrier, support orsurface. For example, the moiety can have affinity to a componentattached to the carrier, support or surface. In this case, the moietymay be, e.g., a biotin or biotinyl group or an analogue thereof bound toan amino acid group of the peptide, such as 6-aminohexanoic acid, andthe component is then avidin, streptavidin, neutravidin, or an analoguethereof. An alternative is a situation in which the moiety has the aminoacid sequence His-His-His-His-His-His (SEQ ID NO: 82) and the carriercomprises a Nitrilotriacetic Acid (NTA) derivative charged with Ni⁺⁺ orCo⁺⁺ ions. In certain embodiments, the moiety is a fusion partner, e.g.,BSA. In exemplary embodiments, peptides of the invention may beconjugated to BSA via N-terminal and/or C-terminal residues of thepeptides. In one embodiment, one, two, three, four, five, 10, 15, 20,25, 30 or more peptides of the invention may be substituted into, e.g.,conjugated with BSA. As would be understood by one skilled in the art,substitution levels may impact the sensitivity of the assay. Lowerconcentrations of highly substituted BSA are needed to achievesensitivity offered by high concentrations of BSA-peptide containingfewer molecules of peptide. In certain other embodiments, the fusionpartner may be MAPS. In certain exemplary embodiments, MAPS may consistof 4, 8, or more asymmetric branches.

Suitable carriers, supports, and surfaces include, but are not limitedto, metallic nanolayers, beads (e.g., magnetic beads, colloidalparticles or metallic nanoparticles or nanoshells, such as colloidalgold, or particles or nanoparticles comprising silica, latex,polystyrene, polycarbonate, or PDVF), latex of co-polymers such asstyrene-divinyl benzene, hydroxylated styrene-divinyl benzene,polystyrene, carboxylated polystyrene, beads of carbon black,non-activated or polystyrene or polyvinyl chloride activated glass,epoxy-activated porous magnetic glass, gelatin or polysaccharideparticles or other protein particles, red blood cells, mono- orpolyclonal antibodies or Fab fragments of such antibodies.

The protocols for immunoassays using antigens for detection of specificantibodies are well known in art. For example, a conventional sandwichassay can be used, or a conventional competitive assay format can beused. For a discussion of some suitable types of assays, see CurrentProtocols in Immunology (supra). In certain embodiments, a peptide ofthe invention is immobilized on a solid or semi-solid surface or carrierby means of covalent or non-covalent binding, either prior to or afterthe addition of the sample containing antibody.

Devices for performing specific binding assays, especially immunoassays,are known and can be readily adapted for use in the present methods.Solid phase assays, in general, are easier to perform than heterogeneousassay methods which require a separation step, such as precipitation,centrifugation, filtration, chromatography, or magnetism, becauseseparation of reagents is faster and simpler. Solid-phase assay devicesinclude microtiter plates, flow-through assay devices (e.g., lateralflow immunoassay devices), dipsticks, and immunocapillary orimmunochromatographic immunoassay devices.

In embodiments of the invention, the solid or semi-solid surface orcarrier is the floor or wall in a microtiter well, a filter surface ormembrane (e.g., a nitrocellulose membrane or a PVDF (polyvinylidenefluoride) membrane, such as an Immobilon™ membrane), a hollow fiber, abeaded chromatographic medium (e.g., an agarose or polyacrylamide gel),a magnetic bead, a fibrous cellulose matrix, an HPLC matrix, an FPLCmatrix, a substance having molecules of such a size that the moleculeswith the peptide bound thereto, when dissolved or dispersed in a liquidphase, can be retained by means of a filter, a substance capable offorming micelles or participating in the formation of micelles allowinga liquid phase to be changed or exchanged without entraining themicelles, a water-soluble polymer, or any other suitable carrier,support or surface.

In some embodiments of the invention, the peptide is provided with asuitable label which enables detection. Conventional labels may be usedwhich are capable, alone or in concert with other compositions orcompounds, of providing a detectable signal. Suitable labels include,but are not limited to, enzymes (e.g., HRP, beta-galactosidase, alkalinephosphatase, etc.), fluorescent labels, radioactive labels, coloredlatex particles, and metal-conjugated labels (e.g., metallic nanolayers,metallic nanoparticle- or metallic nanoshell-conjugated labels).Suitable metallic nanoparticle or metallic nanoshell labels include, butare not limited to, gold particles, silver particles, copper particles,platinum particles, cadmium particles, composite particles, gold hollowspheres, gold-coated silica nanoshells, and silica-coated gold shells.Metallic nanolayers suitable for detectable layers include nanolayerscomprised of cadmium, zinc, mercury, and noble metals, such as gold,silver, copper, and platinum.

Suitable detection methods include, e.g., detection of an agent which istagged, directly or indirectly, with a colorimetric assay (e.g., fordetection of HRP or beta-galactosidase activity), visual inspectionusing light microscopy, immunofluorescence microscopy, includingconfocal microscopy, or by flow cytometry (FACS), autoradiography (e.g.,for detection of a radioactively labeled agent), electron microscopy,immunostaining, subcellular fractionation, or the like. In oneembodiment, a radioactive element (e.g., a radioactive amino acid) isincorporated directly into a peptide chain; in another embodiment, afluorescent label is associated with a peptide via biotin/avidininteraction, association with a fluorescein conjugated antibody, or thelike. In one embodiment, a detectable specific binding partner for theantibody is added to the mixture. For example, the binding partner canbe a detectable secondary antibody or other binding agent (e.g., proteinA, protein G, protein L or combinations thereof) which binds to thefirst antibody. This secondary antibody or other binding agent can belabeled, e.g., with a radioactive, enzymatic, fluorescent, luminescent,metallic nanoparticle or metallic nanoshell (e.g. colloidal gold), orother detectable label, such as an avidin/biotin system. In anotherembodiment, the binding partner is a peptide of the invention, which canbe conjugated directly or indirectly (e.g. via biotin/avidininteraction) to an enzyme, such as horseradish peroxidase or alkalinephosphatase or other signaling moiety. In such embodiments, thedetectable signal is produced by adding a substrate of the enzyme thatproduces a detectable signal, such as a chromogenic, fluorogenic, orchemiluminescent substrate.

A “detection system” for detecting bound peptide, as used herein, maycomprise a detectable binding partner, such as an antibody specific forthe peptide. In one embodiment, the binding partner is labeled directly.In another embodiment, the binding partner is attached to a signalgenerating reagent, such as an enzyme that, in the presence of asuitable substrate, can produce a detectable signal. A surface forimmobilizing the peptide may optionally accompany the detection system.

In some embodiments of the invention, the detection procedure comprisesvisibly inspecting the antibody-peptide complex for a color change, orinspecting the antibody-peptide complex for a physical-chemical change.Physical-chemical changes may occur with oxidation reactions or otherchemical reactions. They may be detected by eye, using aspectrophotometer, or the like.

A particularly useful assay format is a lateral flow immunoassay format.Antibodies to human or animal (e.g., dog, mouse, deer, etc.)immunoglobulins, or staph A, G, or L proteins, can be labeled with asignal generator or reporter (e.g., colloidal gold) that is dried andplaced on a glass fiber pad (sample application pad or conjugate pad).The diagnostic peptide is immobilized on membrane, such asnitrocellulose or a PVDF (polyvinylidene fluoride) membrane (e.g., anImmobilon™ membrane). When a solution of sample (blood, serum, etc.) isapplied to the sample application pad (or flows through the conjugatepad), it dissolves the labeled reporter, which then binds to allantibodies in the sample. The resulting complexes are then transportedinto the next membrane (PVDF or nitrocellulose containing the diagnosticpeptide) by capillary action. If antibodies against the diagnosticpeptide are present, they bind to the diagnostic peptide striped on themembrane, thereby generating a signal (e.g., a band that can be seen orvisualized). An additional antibody specific to the labeled antibody ora second labeled antibody can be used to produce a control signal.

An alternative format for the lateral flow immunoassay comprises thepeptides or compositions of the invention being conjugated to a ligand(e.g., biotin) and complexed with labeled ligand receptor (e.g.,streptavidin-colloidal gold). The labeled peptide complexes can beplaced on the sample application pad or conjugate pad. Anti-humanIgG/IgM or anti-animal (e.g., dog, mouse, deer) IgG/IgM antibodies orother peptides of the invention are immobilized on a membrane, such asnitrocellulose of PVDF, at a test site (e.g., a test line). When sampleis added to the sample application pad, antibodies in the sample reactwith the labeled peptide complexes such that antibodies that bind topeptides of the invention become indirectly labeled. The antibodies inthe sample are then transported into the next membrane (PVDF ornitrocellulose containing the diagnostic peptide) by capillary actionand bind to the immobilized anti-human IgG/IgM or anti-animal IgG/IgMantibodies (or protein A, protein G, protein A/G fusion proteins,protein L, or combinations thereof) or immobilized peptides of theinvention. If any of the sample antibodies are bound to the labeledpeptides of the invention, the label associated with the peptides can beseen or visualized at the test site. Another embodiment of this type oflateral flow device in which the peptides of the invention are used bothas the immobilized capture agent at a test site and as a soluble labeledcomplex to react with antibodies in a sample is shown in FIG. 1. In suchembodiments, to amplify the detection signal, protein A, protein G,and/or protein A/G fusion proteins conjugated to a detectable label(e.g., metallic nanoparticle or nanoshell, HRP, ALP, fluorophore,colored latex particle) may be applied to the test site where they willbind to the Fc region of any antibodies to Ehrlichia antigens capturedby the immobilized peptides of the invention. Suitable controls for thisassay can include, e.g., a chicken IgY-colloidal gold conjugate locatedat the sample application pad or conjugate pad, and an anti-chicken IgYantibody immobilized at a control site located proximal to the testsite.

Another assay for the screening of blood products or other physiologicalor biological fluids is an enzyme linked immunosorbent assay, i.e., anELISA. Typically in an ELISA, isolated peptides or mixtures orpopulations of peptides of the invention are adsorbed to the surface ofa microtiter well directly or through a capture matrix (e.g., anantibody). Residual, non-specific protein-binding sites on the surfaceare then blocked with an appropriate agent, such as bovine serum albumin(BSA), heat-inactivated normal goat serum (NGS), or BLOTTO (a bufferedsolution of nonfat dry milk which also contains a preservative, salts,and an antifoaming agent). The well is then incubated with a biologicalsample suspected of containing specific anti-Ehrlichia (e.g., anti-E.chaffeensis, anti-E. muris, anti-E. ewingii, or anti-E. canis) antibody.The sample can be applied neat, or more often it can be diluted, usuallyin a buffered solution which contains a small amount (0.1-5.0% byweight) of protein, such as BSA, NGS, or BLOTTO. After incubating for asufficient length of time to allow specific binding to occur, the wellis washed to remove unbound protein and then incubated with an optimalconcentration of an appropriate anti-immunoglobulin antibody (e.g., forhuman subjects, an anti-human immunoglobulin (αHuIg) from anotheranimal, such as dog, mouse, cow, etc.) or another peptide of theinvention that is conjugated to an enzyme or other label by standardprocedures and is dissolved in blocking buffer. The label can be chosenfrom a variety of enzymes, including horseradish peroxidase (HRP),beta-galactosidase, alkaline phosphatase (ALP), glucose oxidase, etc.Sufficient time is allowed for specific binding to occur again, then thewell is washed again to remove unbound conjugate, and a suitablesubstrate for the enzyme is added. Color is allowed to develop and theoptical density of the contents of the well is determined visually orinstrumentally (measured at an appropriate wave length). The cutoff ODvalue may be defined as the mean OD+3 standard deviations (SDs) of atleast 50 serum samples collected from individuals from an area whereehrlichiosis is not endemic, or by other such conventional definitions.In the case of a very specific assay, OD+2 SD can be used as a cutoffvalue.

In one embodiment of an ELISA, a peptide of the invention is immobilizedon a surface, such as a ninety-six-well ELISA plate or equivalent solidphase that is coated with streptavidin or an equivalent biotin-bindingcompound, such as avidin or neutravidin, at an optimal concentration inan alkaline coating buffer and incubated at 4° C. overnight. After asuitable number of washes with standard washing buffers, an optimalconcentration of a biotinylated form of a peptide or composition of theinvention, dissolved in a conventional blocking buffer, is applied toeach well. A sample is then added, and the assay proceeds as above.Conditions for performing ELISA assays are well-known in the art.

In another embodiment of an ELISA, a peptide or a mixture of peptides ofthe invention is immobilized on a surface, such as a ninety-six-wellELISA plate or equivalent solid phase via a fusion partner, e.g., BSA orMAPS. A sample is then added and the assay proceeds as above.

An alternative format for the ELISA assay features the peptide(s) of theinvention being attached (e.g., fused) to an appropriate enzyme, such asHRP. Steps for carrying out such an ELISA include: coating the wells ofa plate with anti-dog, anti-cat, or anti-human IgG/IgM; incubatingsamples suspected of containing antibodies to the peptide of theinvention with the immobilized anti-species IgG/IgM; removing unreactedsample and washing the wells with a suitable wash buffer; applyingenzyme-coupled (e.g., HRP-coupled) peptide of the invention and allowingit to react with any captured anti-Ehrlichia antibodies; and visualizingthe enzyme-coupled peptide by applying an appropriate enzyme substrate(e.g., TMB).

In another embodiment, the methods comprise an agglutination assay. Forexample, in certain embodiments, metallic nanoparticles or metallicnanoshells (e.g., colloidal gold, etc.) or latex beads are conjugated topeptides or compositions of the invention. Subsequently, the biologicalfluid is incubated with the bead/peptide conjugate, thereby forming areaction mixture. The reaction mixture is then analyzed to determine thepresence of the antibodies. In certain embodiments, the agglutinationassays comprise the use of a second population of particles, such asmetallic nanoparticles or metallic nanoshells (e.g., colloidal gold,etc.) or latex beads, conjugated to (1) antibodies specific to thepeptides of compositions of the invention, in the case of a competitionassay, or (2) antibodies capable of detecting sample antibodies (e.g.,anti-human IgG or IgM antibodies, anti-dog IgG or IgM antibodies,anti-cat IgG or IgM antibodies, etc.), in the case of a sandwich assay.Suitable agglutination methods can comprise centrifugation as a means ofassessing the extent of agglutination.

In still other embodiments, peptide or compositions of the invention areelectro- or dot-blotted onto nitrocellulose paper. Subsequently, asample, such as a biological fluid (e.g., serum or plasma) is incubatedwith the blotted antigen, and antibody in the biological fluid isallowed to bind to the antigen(s). The bound antibody can then bedetected, e.g., by standard immunoenzymatic methods or by visualizationusing metallic nanoparticles or nanoshells coupled to secondaryantibodies or other antibody binding agents, such as protein A, proteinG, protein A/G fusion proteins, protein L, or combinations thereof.

It should be understood by one of skill in the art that any number ofconventional protein assay formats, particularly immunoassay formats,may be designed to utilize the isolated peptides of this invention forthe detection of Ehrlichia antibodies and infection by pathogenicEhrlichia (e.g., E. chaffeensis, E. muris, E. ewingii, or E. canis) in asubject. This invention is thus not limited by the selection of theparticular assay format, and is believed to encompass assay formats thatare known to those of skill in the art.

In certain embodiments, the sample used in the methods is a bodilyfluid, such as blood, serum, cerebral spinal fluid, urine, or saliva. Inother embodiments, the sample is a tissue (e.g., a tissue homogenate) ora cell lysate. In certain embodiments, the sample is from a wild animal(e.g., a deer or rodent, such as a mouse, chipmunk, squirrel, etc.). Inother embodiments, the sample is from a lab animal (e.g., a mouse, rat,guinea pig, rabbit, monkey, primate, etc.). In other embodiments, thesample is from a domesticated or feral animal (e.g., a dog, a cat, ahorse). In still other embodiments, the sample is from a human.

Much of the preceding discussion is directed to the detection ofantibodies against pathogenic Ehrlichia. However, it is to be understoodthat the discussion also applies to the detection of primed T-cells,either in vitro or in vivo.

It is expected that a cell-mediated immune response (e.g., a T-helperresponse) is generated, since IgG is produced. It is therefore expectedthat it will be possible to determine the immunological reactivitybetween primed T-cells and a peptide of the invention. In vitro this canbe done by incubating T-cells isolated from the subject with a peptideof the invention and measuring the immunoreactivity, e.g., by measuringsubsequent T-cell proliferation or by measuring release of cytokinesfrom the T-cells, such as IFN-γ. These methods are well-known in theart.

When a method of the invention is carried out in vivo, any of a varietyof conventional assays can be used. For example, one can perform anassay in the form of a skin test, e.g., by intradermally injecting, inthe subject, a peptide of the invention. A positive skin reaction at thelocation of injection indicates that the subject has been exposed to andinfected with a pathogenic Ehrlichia capable of causing monocytic and/orgranulocytic ehrlichiosis, and a negative skin response at the locationof injection indicates that the subject has not been soexposed/infected. This or other in vivo tests rely on the detection of aT-cell response in the subject.

In another aspect, the invention provides methods of diagnosingmonocytic and/or granulocytic ehrlichiosis in a subject. The subject canbe a subject suspected of having antibody against a causative agent ofmonocytic and/or granulocytic ehrlichiosis. The diagnostic method isuseful for diagnosing subjects exhibiting the clinical symptoms ofmonocytic and/or granulocytic ehrlichiosis. Clinical symptoms of humanmonocytic/granulocytic ehrlichiosis include, but are not limited to,fever, headache, malaise, myalgia, rash, thrombocytopenia, leukopenia,and elevated serum transaminase levels. Clinical symptoms ofehrlichiosis in animals (e.g. canines) include, but are not limited to,fever, petechiae, bleeding disorders, vasculitis, lymphadenopathy,discharge from the nose and eyes, edema of the legs and scrotum, weightloss, pale gums due to anemia, bleeding due to thrombocytopenia,vasculitis, lymphadenopathy, dyspnea, coughing, polyuria, polydipsia,and lameness.

In certain embodiments, the methods comprise contacting a sample fromthe subject with a peptide of the invention, and detecting formation ofan antibody-peptide complex comprising said peptide, wherein formationof said complex is indicative of the subject having ehrlichiosisdisease. In certain embodiments, the methods comprise contacting thesample with a mixture or population of two, three, four, or more (e.g.,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150,200, 250, 300, 400, 500, or more) different peptides of the invention.For instance, in one particular embodiment, the methods comprisecontacting the sample with a mixture or population of two or moredifferent isolated peptides, wherein each isolated peptide comprises asequence of SEQ ID NO: 1. In another particular embodiment, the methodscomprise contacting the sample with a mixture or population of two ormore different isolated peptides, wherein each isolated peptidecomprises a sequence of SEQ ID NO: 3. In still another embodiment, themethods comprise contacting the sample with a mixture or population oftwo or more different isolated peptides, wherein each isolated peptidecomprises a sequence of SEQ ID NO: 72. In some embodiments, the methodscomprise contacting the sample with a mixture or population of two ormore different isolated peptides, wherein each isolated peptidecomprises a sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 72. Inother embodiments, the methods comprise contacting the sample with amixture or population of two or more different isolated peptides,wherein each isolated peptide comprises a sequence of SEQ ID NO: 5, SEQID NO: 7, SEQ ID NO: 70, SEQ ID NO: 83, or SEQ ID NO: 85.

In certain embodiments, the methods comprise contacting the sample witha mixture of one or more peptides or the invention and one or more otherpeptides (e.g., an Ehrlichia peptide, or antigenic fragment or epitopethereof, such as from an Ehrlichia surface protein or an EhrlichiaOMP-1, p38, p43, p120, p140, p153, p156, p200, gp19, gp36, gp47, gp200,or HGE-3 protein. For instance, in some embodiments, the methodscomprise contacting the sample with a mixture of one or more peptides ofthe invention (e.g., SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 83, or SEQ ID NO: 85)and one or more Ehrlichia antigenic peptides having a sequence of SEQ IDNO: 71. In one particular embodiment, the methods comprise contactingthe sample with a mixture of one or more peptides having a sequence ofSEQ ID NO: 3 and one or more peptides having a sequence of SEQ ID NO:71. In another embodiment, the methods comprise contacting the samplewith a mixture of one or more peptides having a sequence of SEQ ID NO:72 and one or more peptides having a sequence of SEQ ID NO: 71. Inanother embodiment, the methods comprise contacting the sample with amixture of one or more peptides having a sequence of SEQ ID NO: 72 andone or more peptides having a sequence of SEQ ID NO: 3. In oneembodiment, the methods comprise contacting the sample with a mixture ofone or more peptides having a sequence of SEQ ID NO: 72, one or morepeptides having a sequence of SEQ ID NO: 3, and one or more peptideshaving a sequence of SEQ ID NO: 71.

In certain embodiments, the peptide or each peptide in the mixture orpopulation is an isolated (e.g., synthetic and/or purified) peptide. Incertain embodiments, the peptide or mixture of different peptides (i.e.population of peptides) is attached to or immobilized upon a substrate(e.g., a solid or semi-solid support). For example, in certainembodiments, the substrate is a bead (e.g., a colloidal or other type ofparticle or metallic nanoparticle or nanoshell), a flow path in alateral flow immunoassay device (e.g., a porous membrane), a flow pathin an analytical or centrifugal rotor, a blot (e.g., a Western blot, dotblot, or slot blot), a tube or a well (e.g., in a plate suitable for anELISA assay), or a sensor (e.g., an electrochemical, optical, oropto-electronic sensor). In some embodiments, the peptide or mixture ofpeptides is attached to or immobilized upon a solid support through ametallic nanolayer that, in some embodiments, may be comprised ofcadmium, zinc, mercury, or a noble metal (e.g., gold, silver, copper,and platinum).

There are a number of different conventional assays for detectingformation of an antibody-peptide complex comprising a peptide of theinvention. For example, the detecting step can comprise performing anELISA assay, performing a lateral flow immunoassay, performing anagglutination assay, performing a wavelength shift assay, analyzing thesample using a Western blot, a slot blot, or a dot blot, analyzing thesample in an analytical or centrifugal rotor, or analyzing the samplewith an electrochemical, optical, or opto-electronic sensor. Thesedifferent assays are described above and/or are well-known to thoseskilled in the art.

In certain embodiments, the sample is a bodily fluid, such as blood,serum, cerebral spinal fluid, urine, or saliva. In other embodiments,the sample is a tissue (e.g., a tissue homogenate) or a cell lysate. Incertain embodiments, the subject is a wild animal (e.g., a deer orrodent, such as a mouse, chipmunk, squirrel, etc.). In otherembodiments, the subject is a lab animal (e.g., a mouse, rat, guineapig, rabbit, monkey, primate, etc.). In other embodiments, the subjectis a domesticated or feral animal (e.g., a dog, a cat, a horse). Instill other embodiments, the subject is a human.

Kits

In yet another aspect, the invention provides kits. In certainembodiments, the kits comprise a peptide of the invention. In certainembodiments, the kits comprise two, three, four, or more differentpeptides of the invention or a mixture or population of the peptides ofthe invention. The peptides can comprise a sequence of SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 7, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 83, SEQID NO: 85, or fragments thereof. In certain embodiments, the peptidesare attached to or immobilized on a solid support. In some embodiments,the peptides are attached to or immobilized on a solid support through ametallic nanolayer (e.g., cadmium, zinc, mercury, gold, silver, copper,or platinum nanolayer). In certain embodiments, the solid support is abead (e.g., a colloidal particle or a metallic nanoparticle ornanoshell), a flow path in a lateral flow immunoassay device, a flowpath in an analytical or centrifugal rotor, a tube or a well (e.g., in aplate), or a sensor (e.g., an electrochemical, optical, oropto-electronic sensor).

Reagents for particular types of assays can also be provided in kits ofthe invention. Thus, the kits can include a population of beads (e.g.,suitable for an agglutination assay or a lateral flow assay), or a plate(e.g., a plate suitable for an ELISA assay). In other embodiments, thekits comprise a device, such as a lateral flow immunoassay device, ananalytical or centrifugal rotor, a Western blot, a dot blot, a slotblot, or an electrochemical, optical, or opto-electronic sensor. Thepopulation of beads, the plate, and the devices are useful forperforming an immunoassay. For example, they can be useful for detectingformation of an antibody-peptide complex comprising an antibody from asample and a peptide of the invention. In certain embodiments, apeptide, a mixture of different peptides (i.e. population of peptides)of the invention, or a peptide composition of the invention is attachedto or immobilized on the beads, the plate, or the device.

In addition, the kits can include various diluents and buffers, labeledconjugates or other agents for the detection of specifically boundantigens or antibodies (e.g. labeling reagents), and othersignal-generating reagents, such as enzyme substrates, cofactors andchromogens. In some embodiments, the kit comprises an anti-human,anti-canine, or anti-feline IgG/IgM antibody conjugated to a detectablelabel (e.g., a metallic nanoparticle, metallic nanoshell, metallicnanolayer, fluorophore, colored latex particle, or enzyme) as a labelingreagent. In other embodiments, the kit comprises protein A, protein G,protein A/G fusion proteins, protein L, or combinations thereofconjugated to a detectable label (e.g., a metallic nanoparticle,metallic nanoshell, metallic nanolayer, fluorophore, colored latexparticle, or enzyme) as a labeling reagent. An exemplary protein A/Gfusion protein combines four Fc-binding domains from protein A with twofrom protein G. See, e.g., Sikkema, J. W. D., Amer. Biotech. Lab, 7:42,1989 and Eliasson et al., J. Biol. Chem. 263, 4323-4327, 1988, bothwhich are hereby incorporated by reference in their entireties. In stillother embodiments, the labeling reagents of the kit are a secondpopulation of peptides of the invention conjugated to a detectable label(e.g., a metallic nanoparticle, metallic nanoshell, metallic nanolayer,fluorophore, colored latex particle, or enzyme). The second populationof peptides can be the same as or different than the first population ofpeptides, which may optionally be attached to or immobilized upon asolid support.

Other components of a kit can easily be determined by one of skill inthe art. Such components may include coating reagents, polyclonal ormonoclonal capture antibodies specific for a peptide of the invention,or a cocktail of two or more of the antibodies, purified orsemi-purified extracts of these antigens as standards, monoclonalantibody detector antibodies, an anti-mouse, anti-dog, anti-cat,anti-chicken, or anti-human antibody conjugated to a detectable label,indicator charts for colorimetric comparisons, disposable gloves,decontamination instructions, applicator sticks or containers, a samplepreparatory cup, etc. In one embodiment, a kit comprises buffers orother reagents appropriate for constituting a reaction medium allowingthe formation of a peptide-antibody complex.

Such kits provide a convenient, efficient way for a clinical laboratoryto diagnose infection by a pathogenic Ehrlichia, such as E. chaffeensis,E. muris, E. ewingii, or E. canis. Thus, in certain embodiments, thekits further comprise an instruction. For example, in certainembodiments, the kits comprise an instruction indicating how to use apeptide or population of peptides of the invention to detect an antibodyto one or more Ehrlichia antigens or to diagnose monocytic and/orgranulocytic ehrlichiosis. In certain embodiments, the kits comprise aninstruction indicating how to use a population of beads, a plate, or adevice (e.g., comprising a peptide or a mixture of different peptides ofthe invention) to detect an antibody to one or more Ehrlichia antigensor to diagnose monocytic and/or granulocytic ehrlichiosis.

The peptides, compositions and devices comprising the peptides, kits andmethods of the invention offer a number of advantages. For example, theyallow for simple, inexpensive, rapid, sensitive and accurate detectionof monocytic and/or granulocytic ehrlichiosis, and avoid serologiccross-reactivity with other conditions with similar symptoms. Thisallows for an accurate diagnosis. Furthermore, a diagnostic test of theinvention (e.g., an ELISA assay, lateral flow immunoassay, oragglutination assay) is useful in serum samples that contain anti-OMP-1antibodies or other antibodies produced in response to a vaccine basedon the outer surface proteins of Ehrlichia.

The following examples illustrate various aspects of the invention. Theexamples should, of course, be understood to be merely illustrative ofonly certain embodiments of the invention and not to constitutelimitations upon the scope of the invention.

EXAMPLES Example 1 ELISA Assay

Three different populations of peptides were synthesized using standardsynthesis procedures. Each peptide in the first population of peptides(ECHEW1) contained a sequence of SEQ ID NO: 72. The first population ofpeptides specifically binds to antibodies elicited by multiple Ehrlichiaspp. (e.g., canis, chaffeensis, and ewingii). Each peptide in the secondpopulation of peptides (EE12EW1) contained a sequence of SEQ ID NO: 3.The second population of peptides specifically binds to antibodieselicited primarily by E. canis and E. chaffeensis with somecross-reactivity to E. ewingii. Each peptide in the third population ofpeptides (EE13) contained a sequence of SEQ ID NO: 71. The thirdpopulation of peptides specifically binds to antibodies elicitedprimarily by E. ewingii with some cross-reactivity to E. canis and E.chaffeensis.

Each peptide in the three populations was linked separately to thecarrier protein bovine serum albumin (BSA) using thio-ether chemistry.The resulting BSA-peptide conjugates were used as capture entities in96-well ELISA plates to create three separate ELISA assays (onepopulation of peptides per plate). The plates were then blocked toprevent undesirable non-specific binding.

Dog plasma samples positive to Ehrlichia species, as determined byindirect immunofluorescence assays (IFA), IDEXX SNAP 4DX Plus™, and/orSNAP 3Dx™, were incubated with the immobilized capture peptides in eachof the three ELISA plates. After one hour incubation, the unreactedmaterials were removed by washing the micro wells. The specificallycaptured dog IgG or IgM were detected by reaction with HRP-labeledProtein A. HRP was assayed using a commercial TMB substrate. The opticaldensity of each well was read at 650 nm with a plate reader.

A total of 156 samples were evaluated, of which 152 tested positive inthe ELISA plates with four samples testing negative. Thus, the percentsensitivity of the test was 97.4%. A summary of the results separated byinfective Ehrlichia species is shown in Table 1 below. Theseexperimental results show that populations of peptides defined by SEQ IDNO: 72, SEQ ID NO: 3, or SEQ ID NO: 71 have a high degree of sensitivityin detecting the presence of antibodies to antigens from variousEhrlichia spp.

TABLE 1 ELISA Results of Known Ehrlichia-Positive Samples Positive byELISA Negative by ELISA Total No. of E. canis- 44 1 45 positive samples¹No. of E. chaffeensis- 38 2 40 positive samples² No. of E. ewingii- 46 147 positive samples³ No. of positive 24 0 24 samples; indeterminatespecies⁴ Total Samples Tested 156 Total No. of Positive 152 SamplesCorrectly Identified (detected positive by ELISA) Total No. of Positive4 Samples Incorrectly Identified (detected negative by ELISA) %Sensitivity 97.4 ¹These samples tested positive in the ELISA assays withECHEW1 (SEQ ID NO: 72) and EE12EW1 (SEQ ID NO: 3) populations ofpeptides and had a higher titer for E. canis by IFA. ²These samplestested positive in the ELISA assays with ECHEW1 (SEQ ID NO: 72) andEE12EW1 (SEQ ID NO: 3) populations of peptides and had a higher titerfor E. chaffeensis by IFA. ³These samples tested positive in the ELISAassays with ECHEW1 (SEQ ID NO: 72) and EE13 (SEQ ID NO: 71) populationsof peptides. ⁴The species of Ehrlichia in these samples could not bedetermined conclusively by IFA or the SNAP assays, but the samplestested positive in the ELISA assay with ECHEW1 (SEQ ID NO: 72).

Example 2 Lateral Flow Assay

A lateral flow immunoassay in a double antigen sandwich format wasconstructed to detect the presence of antibodies specific for Ehrlichiaantigens from multiple species. A population of peptides defined by SEQID NO: 72 was linked to BSA and the resulting complexes were used bothas test conjugate (peptides labeled with gold nanoparticles) and ascapture (immobilized at the test line of the device). The signalproduced at the test line was enhanced by Protein A and Protein G-goldconjugates added to the labeled peptide conjugate. The device isdepicted in FIG. 5.

To conduct the assay, one drop of anti-coagulated whole blood, serum, orplasma is applied to the sample port of the device. The blood separationpad filters blood cells from whole blood. Plasma (or serum) mobilizesand binds specifically to the test conjugate present on the conjugatepad and any formed antibody-peptide complexes migrate to thenitrocellulose membrane containing the test and the control regions. Theapplication of a chase buffer after sample application moves the freeand the bound test conjugates through the nitrocellulose membranetowards the upper absorbent pad. The labeled peptide-antibody complexesmove to the test line where immobilized peptides capture labeledpeptide-antibody complexes via the second binding sites on theantibodies. Protein A-gold and Protein G-gold conjugates in theconjugate mixture bind to captured antibodies amplifying the detectionsignal. The appearance of one red line at the test site and a second redline at the control site indicates the presence of antibodies toEhrlichia spp. (e.g., canis, chaffeensis, or ewingii) in the sample. Theappearance of a red line at only the control site indicates the absenceof antibodies to all of the Ehrlichia spp. in the sample. The test isconsidered invalid if (i) a signal at the test line appears but nosignal at the control line is present or (ii) no signal is observed ateither the control or test lines.

The same 156 known Ehrlichia-positive dog plasma samples evaluated byELISA assay in Example 1 were tested in the lateral flow device. Inaddition, 120 dog samples (100 plasma and 20 whole blood samples) thatwere determined to be negative by indirect immunofluorescence assays orIDEXX SNAP 4DX Plus™, were also evaluated. The results are summarized inTable 2 below. The lateral flow assay had a sensitivity of 97.4% with a95% confidence interval of 93.6-99.3%. The specificity of the assay was98.3% with a 95% confidence interval of 94.1-99.8%. This exampledemonstrates that a population of peptides defined by SEQ ID NO: 72 caneffectively detect antibodies against Ehrlichia antigens when employedin a lateral assay format.

TABLE 2 Lateral Flow Assay Results of Known Ehrlichia-Positive andNegative Samples Negative by Positive by Lateral Flow Lateral Flow No.of known negative 118 2 samples No. of known positive 4 152 samples

Example 3 Indirect Fluorescent Antibody Assay

An indirect fluorescent antibody test is constructed using latex beadscoated with one or more peptides of the invention. In certainembodiments, the peptides defined by SEQ ID NO: 71, SEQ ID NO: 72, SEQID NO: 3, or SEQ ID NO: 5 are used. The peptides of the invention arecoated onto maleimide-derivatized latex beads using thio-etherchemistry. Alternatively, the peptides of the invention may beconjugated to BSA via thio-ether or similar chemistries and arepassively absorbed on to latex beads. A population of such beads is thenimmobilized on a glass slide using known techniques.

To conduct the assay, one drop of serum or plasma (diluted appropriatelywith a suitable buffer) from dogs suspected of having anti-Ehrlichiaantibodies, is applied to the glass slide coated with latex beads.Following a suitable incubation time, the unreacted materials are washedaway and a drop of fluorescently labeled anti-dog IgG (or IgM) isapplied and the slides are incubated for an additional time period. Thefinal preparation is viewed under a fluorescent microscope to determinefluorescently tagged latex beads. The classification of the testserum/plasma as positive or negative is based on comparison withappropriate controls. An enzyme label may be used in place of thefluorescent label in which case the visualization step employs an enzymesubstrate. For example, anti-dog IgG/IgM labeled with alkalinephosphatase can be visualized by exposing the slide to a BCIP-nitro BTsubstrate. Labeled Protein A, Protein G, or Protein A/G fusion can beused in place of labeled anti-dog IgG and anti-dog IgM to detectantibodies bound to the peptide-coated beads.

To the extent that any definitions in documents incorporated byreference are inconsistent with the definitions provided herein, thedefinitions provided herein are controlling. Although the invention hasbeen described with reference to the presently preferred embodiments, itshould be understood that various changes and modifications, as would beobvious to one skilled in the art, can be made without departing fromthe spirit of the invention. Accordingly, the invention is limited onlyby the following claims.

The disclosures, including the claims, figures and/or drawings, of eachand every patent, patent application, and publication cited herein arehereby incorporated herein by reference in their entireties.

What is claimed:
 1. A population of isolated peptides comprising threeor more different peptides, wherein each peptide in the populationcomprises a sequence of: (i)S-X₂-K-E-X₅-K-Q-X₈-T-X₁₀-X₁₁-X₁₂-X₁₃-G-L-K-Q-X₁₈-W-X₂₀-G-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-G-G-G-G-G-N-F-S-A-K-E-E-X₃₉-A-E-T-R-X₄₄-T-F-G-L-X₄₉-K-Q-Y-D-G-A-X₅₆-I-X₅₈-E-N-Q-V-Q-N-K-F-T-I-S-N-C(SEQ ID NO: 72) or fragment thereof, wherein X₂ is an amino acidselected from the group consisting of A and V, X₅ is an amino acidselected from the group consisting of E and D, X₈ is an amino acidselected from the group consisting of T and P, X₁₀ is an amino acidselected from the group consisting of T and V, X₁₁ is an amino acidselected from the group consisting of G and A, X₁₂ is an amino acidselected from the group consisting of L and V, X₁₃ is an amino acidselected from the group consisting of Y and F, X₁₈ is an amino acidselected from the group consisting of D and N, X₂₀ is an amino acidselected from the group consisting of D and N, X₂₂ is an amino acidselected from the group consisting of S and V, X₂₃ is an amino acidselected from the group consisting of A, S, and T, X₂₄ is an amino acidselected from the group consisting of A and I, X₂₅ is an amino acidselected from the group consisting of T and P, X₂₆ is an amino acidselected from the group consisting of S, N, and K, X₃₉ is any aminoacid, X₄₄ is any amino acid, X₄₉ is any amino acid, X₅₆ is any aminoacid, and X₅₈ is any amino acid, or (ii)S-X₂-K-E-X₅-K-Q-X₈-T-X₁₀-X₁₁-X₁₂-X₁₃-G-L-K-Q-X₁₈-W-X₂₀-G-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-G-G-G-G-G-N-F-S-A-K-E-E-X₃₉-A-X₄₁-T-R-X₄₄-T-F-G-X₄₈-X₄₉-K-Q-Y-D-G-A-X₅₆-I-X₅₈-E-N-Q-V-Q-N-K-F-T-I-S-N-C(SEQ ID NO: 3) or a fragment thereof, wherein X₂ is an amino acidselected from the group consisting of A and V, X₅ is an amino acidselected from the group consisting of E and D, X₈ is an amino acidselected from the group consisting of T and P, X₁₀ is an amino acidselected from the group consisting of T and V, X₁₁ is an amino acidselected from the group consisting of G and A, X₁₂ is an amino acidselected from the group consisting of L and V, X₁₃ is an amino acidselected from the group consisting of Y and F, X₁₈ is an amino acidselected from the group consisting of D and N, X₂₀ is an amino acidselected from the group consisting of D and N, X₂₂ is an amino acidselected from the group consisting of S and V, X₂₃ is an amino acidselected from the group consisting of A, S, and T, X₂₄ is an amino acidselected from the group consisting of A and I, X₂₅ is an amino acidselected from the group consisting of T and P, X₂₆ is an amino acidselected from the group consisting of S, N, and K, X₃₉ is any aminoacid, X₄₁ is an amino acid selected from the group consisting of D andN, X₄₄ is any amino acid, X₄₈ is an amino acid selected from the groupconsisting of V and A, X₄₉ is any amino acid, X₅₆ is any amino acid, andX₅₈ is any amino acid.
 2. The population of isolated peptides of claim1, wherein X₃₉ is K.
 3. The population of isolated peptides of claim 1,wherein X₄₄ is an amino acid selected from the group consisting of K andR, and X₄₉ is an amino acid selected from the group consisting of E andD.
 4. The population of isolated peptides of claim 1, wherein X₅₆ is anamino acid selected from the group consisting of K and Q, and X₅₈ is anamino acid selected from the group consisting of E and T.
 5. Thepopulation of isolated peptides of claim 1, wherein said fragmentcomprises at least 20, 25, 30, 35, or 40 contiguous amino acids from SEQID NO: 3 or SEQ ID NO:
 72. 6. The population of isolated peptides ofclaim 5, wherein said fragment comprises amino acids 33 to 71 of SEQ IDNO: 3 or SEQ ID NO:
 72. 7. The population of isolated peptides of claim1, wherein each isolated peptide comprises an additional N-terminalpeptide sequence that is a native OMP-1 sequence or non-OMP-1 Ehrlichiaantigen.
 8. The population of isolated peptides of claim 1, wherein eachisolated peptide comprises an additional C-terminal peptide sequencethat is a native OMP-1 sequence or non-OMP-1 Ehrlichia antigen.
 9. Thepopulation of isolated peptides of claim 1, wherein each isolatedpeptide comprises at least 70, 75, 80, or 85 amino acids.
 10. Thepopulation of isolated peptides of claim 1, wherein one or more of theisolated peptides is conjugated to a ligand or is biotinylated.
 11. Thepopulation of isolated peptides of claim 1, wherein one or more of theisolated peptides is conjugated to avidin, streptavidin, neutravidin,serum albumin, keyhole limpet hemocyanin (KLH), an enzyme, or a metallicnanoparticle or nanoshell.
 12. The population of isolated peptides ofclaim 1, wherein the population of peptides is immobilized to a solidsupport optionally through a metallic nanolayer.
 13. The population ofisolated peptides of claim 1, wherein the solid support is a pluralityof beads, a flow path in a lateral flow immunoassay device, a well in amicrotiter plate, or a flow path in a rotor.
 14. The population ofisolated peptides of claim 1, wherein the population further comprisesone or more antigenic peptides from an Ehrlichia species.
 15. Thepopulation of isolated peptides of claim 14, wherein said one or moreantigenic peptides has a sequence ofF-S-A-K-E-E-X₇-A-E-T-R-X₁₂-T-F-G-L-X₁₇-K-Q-Y-D-G-A-X₂₄-I-X₂₆-E-N-Q-V-Q-N-K-F-T-I-S-N-C(SEQ ID NO: 71) or a fragment thereof, wherein X₇ is any amino acid, X₁₂is any amino acid, X₁₇ is any amino acid, X₂₄ is any amino acid, and X₂₆is any amino acid.
 16. The population of isolated peptides of claim 15,wherein X₇ in SEQ ID NO: 71 is K.
 17. The population of isolatedpeptides of claim 15, wherein X₁₂ in SEQ ID NO: 71 is an amino acidselected from the group consisting of K and R, and X₁₇ in SEQ ID NO: 71is an amino acid selected from the group consisting of E and D.
 18. Thepopulation of isolated peptides of claim 15, wherein X₂₄ in SEQ ID NO:71 is an amino acid selected from the group consisting of K and Q, andX₂₆ in SEQ ID NO: 71 is an amino acid selected from the group consistingof E and T.
 19. A method for detecting in a sample an antibody to anepitope of an Ehrlichia antigen, the method comprising: contacting asample with the population of isolated peptides of claim 1; anddetecting formation of an antibody-peptide complex comprising said oneor more peptides in the population, wherein formation of said complex isindicative of an antibody to an epitope of an Ehrlichia antigen beingpresent in said sample.
 20. The method of claim 19, wherein saidEhrlichia antigen is from an Ehrlichia chaffeensis, Ehrlichia ewingii,Ehrlichia canis, or Ehrlichia muris species.
 21. The method of claim 19,wherein the population of isolated peptides is immobilized to a solidsupport optionally through a metallic nanolayer.
 22. The method of claim21, wherein said solid support is a plurality of beads, a flow path in alateral flow assay device, a well in a microtiter plate, or a flow pathin a rotor.
 23. The method of claim 19, wherein said detecting stepcomprises (i) performing an ELISA assay, (ii) running a lateral flowassay, (iii) performing an agglutination assay, (iv) performing aWestern blot, a slot blot, or dot blot, (v) performing a wavelengthshift assay, or (vi) running the sample through an analytical orcentrifugal rotor.
 24. The method of claim 19, wherein said sample isfrom a human, canine, or feline subject.
 25. The method of claim 19,wherein said sample is a blood, serum, plasma, cerebral spinal fluid,tissue extract, urine, or saliva sample.
 26. The method of claim 19,wherein the formation of a plurality of antibody-peptide complexes aredetected, and wherein formation of said complexes is indicative ofantibodies to epitopes of Ehrlichia antigens from multiple Ehrlichiaspecies being present in said sample.
 27. A method for diagnosingmonocytic and/or granulocytic ehrlichiosis in a subject, the methodcomprising: contacting a sample from the subject with the population ofisolated peptides of claim 1; and detecting formation of anantibody-peptide complex comprising said one or more peptides in thepopulation, wherein formation of the complex is indicative the subjecthaving monocytic and/or granulocytic ehrlichiosis.
 28. A kit comprisingthe population of isolated peptides of claim 1 and a labeling reagentcapable of binding to an antibody that recognizes an epitope of said oneor more peptides in the population.
 29. The kit of claim 28, wherein thepopulation of isolated peptides is attached to or immobilized on a solidsupport optionally through a metallic nanolayer.
 30. The kit of claim28, wherein the solid support is a plurality of beads, a tube or a well,a lateral flow assay device, or an analytical or centrifugal rotor. 31.The kit of claim 28, wherein the labeling reagent is an anti-human,anti-canine, or anti-feline IgG or IgM antibody conjugated to adetectable label.
 32. The kit of claim 31, wherein the detectable labelis an enzyme, a metallic nanoparticle, metallic nanoshell, metallicnanolayer, fluorophore, or colored latex particle.
 33. The kit of claim28, wherein the labeling reagent is protein A, protein G, and/or aprotein A/G fusion protein conjugated to a detectable label.
 34. The kitof claim 33, wherein the detectable label is an enzyme, a metallicnanoparticle, metallic nanoshell, metallic nanolayer, fluorophore, orcolored latex particle.